Team Sky finished in 14th place, 28 seconds behind winners HTC-Columbia, in the floodlit team time trial which opened the 75th anniversary of the Vuelta a España.
Juan Antonio Flecha and Simon Gerrans led them across the line under the lights in Seville along with Peter Kennaugh, Ben Swift and Lars-Petter Nordhaug in a time of 14 minutes and 34 seconds for the 13 kilometres course.
They had to battle back after leading general classification hope Thomas Löfkvist lost touch in the early stages.
Team Principal Dave Brailsford explained: "They went off like a train and Thomas just got distanced a little bit on one of the chicanes. That meant he had to fight really hard to get back which put him into the red and it was hard going for him from then on."
Kjell Carlström dropped back to shepherd Löfkvist home and the pair crossed the line 47 seconds behind Team Sky's leading quintet.
Brailsford added: "Marcus (Ljungqvist, the 1st DS) made the call to wait for Thomas - he did everything he could to get back on but it was tough for him.
"Once your rhythm has been disturbed it's hard to get it back but these things happen. It's not the result we were looking for but at least there's a reason behind it and they'd have been a lot higher up the leaderboard with a smoother run.
"There are a lot of good legs in there - those front guys were flying - but these Grand Tour prologues continue to be pretty dramatic for us; we got the jersey at the Giro with Bradley (Wiggins), we then had the rain at the Tour de France and now this."
Cavendish in leader's red jersey
The final Grand Tour of the season had started at 10.04pm local time and ended not long before midnight but there were no hard luck stories in terms of the weather, with all the teams having near-identical conditions as it was warm and dry throughout.
Team Sky went out at 10.48pm but by then HTC-Columbia had set a mark of 14:06 which never really looked like being threatened and Britain's Mark Cavendish is the first holder of the leader's red jersey as he crossed the line first.
Liquigas came closest to challenging HTC-Columbia and took second, 10 seconds behind, with Cervélo a further three seconds back in third.
Large crowds had lined the route under the floodlights and Brailsford said of the dramatic opening to the Vuelta: "It's good to see cycling trying to do things slightly differently every now and then when the opportunity arises."
Sunday's 173.7km second stage takes the riders from Alcalá de Guadaíra to Marbella and has every chance of ending with a sprint finish.