Ross Barkley missed a late VAR-awarded penalty as Frank Lampard lost 1-0 to Valencia on his Champions League debut as Chelsea manager.
Chelsea had the chance to cancel out Rodrigo's 74th-minute opener and salvage a point when the Video Assistant Referee alerted the on-field official to Daniel Wass' handball in the area with three minutes remaining.
Barkley insisted he should take the spot-kick, snatching the ball off regular penalty taker Jorginho, but his effort cannoned off the top of the crossbar.
- Mason Mount injured on CL debut
- Champions League Group H standings
- Champions League scores and fixtures
The late penalty controversy compounded a harsh introduction to Champions League football for Lampard, who lost Mason Mount to injury just 16 minutes and still awaits the first home victory of his tenure at Chelsea.
Valencia make Chelsea pay the penalty
Valencia players and fans supposedly arrived in London in disarray following the sacking of manager Marcelino last week, but they were united as they produced a classy display against Lampard's youthful side which featured four competition debutants.
Tammy Abraham, fresh from his hat-trick at Wolves, was denied a maiden Champions League goal when his near-post flick was blocked by Valencia goalkeeper Jasper Cillessen.
Mason Mount then received a painful introduction to European football when former Arsenal midfielder Frances Coquelin left his studs on the inside of his shin with a challenge that yielded only a yellow card, despite forcing the youngster off inside 16 minutes.
Willian was Chelsea's biggest threat in the first half and nearly scored a sensational opener when he brought a cross down on his chest and flashed a first-time effort inches over.
Marcos Alonso took the game to Valencia early in the second half, drilling in a free-kick under the visitors' poor excuse for a wall which Cillessen tipped behind for a corner, from which the left-back headed over at the near post.
But with 16 minutes remaining, Valencia struck with their first effort on target when Rodrigo gave the Chelsea defence the slip, raced into acres of space in the Chelsea area and drilled a shot into the group that flew past Kepa Arrizabalaga into the top corner.
Chelsea's players appealed for a handball with five minutes remaining in a dramatic finale, as replays showed Fikayo Tomori's header was blocked by the hand of Valencia defender Wass.
After a lengthy VAR review, a penalty was awarded but Barkley, on as a substitute just seven minutes prior, took responsibility into his own hands only to crash his effort against the bar and over.
What Lampard said
Chelsea manager Frank Lampard: "Ross is the penalty taker. He was in pre-season and scored a couple, has been when he has started, and was today when he came on and he missed it. I don't know what the conversation was. Jorginho and Willian were the penalty takers on the pitch, but when Ross comes on, he becomes the penalty taker.
"We probably deserved a win, or at least a draw. We created enough in the game. Not lots of fantastic chances but good chances and obviously a penalty, then we concede from the one shot on target, so it's a harsh lesson in Champions League football. It's a game we played pretty well in, and we lost.
"The good news is we've got five more games to go in the group, but that's the reality of losing the first game at home, it makes those [other] games more important particularly as they come back to back away from home."
Who's the penalty taker?
Former Chelsea midfielder Steve Sidwell told The Debate:
"It's the same rigmarole as the Paul Pogba one - who's the designated taker? Barkley came on after 80 minutes, Jorginho's on and normally takes them and seems very confident taking penalties. I can't see him not wanting to take one.
"I can see what Willian's doing in terms of saying 'Do you fancy this? You've just come on'. When Barkley says yes, he walks away but he had gone once, twice, three times asking him.
"Before every game, you know who the designated player is. If they're on the pitch, they're taking it. If they want to shift the responsibility, then so be it, but we didn't see that from Jorginho so it's surprising."
- Ross Barkley was the first Englishman to take a penalty for Chelsea since Frank Lampard in April 2014 against Stoke City - both players missed their penalties.
- This was only Chelsea's second defeat in their last 42 Champions League group stage games at Stamford Bridge (W30 D10 L2), with the other against Basel in 2013.
- Valencia registered only their second away Champions League win in England (W2 D7 L3), with their first since winning 1-0 at Liverpool in October 2002.
Live Renault Super Sunday
Chelsea host league leaders Liverpool on Renault Super Sunday - live from 4pm on Sky Sports Premier League; kick-off 4.30pm. Chelsea's next Champions League tie is away to Lille on Matchday 2 of the Champions League on October 2; Kick-off 8pm.