Discovery Channel's George Hincapie powered to a surprise win in the 7.9km prologue on day one of the Dauphine Libere in Aix-Les-Bains on Sunday.
Hincapie is Armstrong's closest friend in the peloton
The New Yorker clocked nine minutes 55 seconds to beat compatriot Gerolsteiner rider Levi Leipheimer by one second.
Kazakhstan's Andryi Kashechkin of the Credit Agricole team was third, three seconds off Hincapie's blistering pace.
Hincapie's team leader Lance Armstrong, who is preparing to go for a seventh Tour de France title, finished fifth.
"I'm not really specialized in prologue any more," said the 33-year-old Armstrong.
"Today I was conservative in the climb, and then there was a lot of headwind in the finish. But that's OK.
"It's a short explosive effort and when you get old, you know what happens. In the last few years I didn't do good prologues. The prologue of the last Tour was an exception. I had a super day that day."
Hincapie, a close friend of Armstrong, said: "I gave everything to the point that I felt like vomiting once I crossed the line.
"It was the first time I have beaten Lance in a time-trial. It's very special."
Earlier, Spain's Isidro Nozal and Italian Michele Scotto d'Abusco were banned from starting the eight-day stage race.
Blood tests showed the pair had haematocrit levels above the legal 50% level and they were automatically suspended.
Nozal of the Liberty Seguros team and Lampre's Scotto d'Abusco failed tests carried out by cycling's governing body, the International Cycling Union, and under new regulations could now face the sack.
Armstrong has won the traditional Tour warm-up twice before.
But he ended the opening day six seconds off the time set by Hincapie - who is not regarded as a time-trial specialist - and faces stiff opposition from some key rivals over the remaining week.
They include last year's Tour runner-up, Andreas Kloden, and Alexander Vinokourov.
But that duo's team leader - and the man marked out by Armstrong himself as his chief Tour rival - Jan Ullrich, will be absent.
Other rivals from Sunday will include CSC rider Bobby Julich, who has been in supreme form this season, and Floyd Landis, Armstrong's former team-mate.
Armstrong is using the event solely to prepare for the Tour - his last race before he retires.
"There are no guarantees that I'll win, but I can tell you that I'm more excited than ever to race," said the Texan.
"For me, to win a final Tour and then be able to stop immediately after is a dream."
The Dauphine Libere includes mountain-top finishes at Mount Ventoux and Joux Plane as well as a 46.5km time trial.
Prologue results (Sun):
1. George Hincapie (United States / Discovery Channel) nine hours 55 minutes
2. Levi Leipheimer (United States / Gerolsteiner) +1 second
3. Andrey Kashechkin (Kazakhstan / Credit Agricole) +3
4. Floyd Landis (United States / Phonak) +5
5. Lance Armstrong (United States / Discovery Channel) +6
6. Oscar Pereiro (Spain / Phonak) +7
7. Alberto Contador (Spain / Liberty Seguros) +9
8. Davide Rebellin (Italy / Gerolsteiner) +13
9. Jose Ivan Gutierrez (Spain / Illes Balears) +13
10. Francisco Jose Lara (Spain / T-Mobile) +14
Dauphine Libere stages:
June 5: (prologue): Aix-les-Bains individual time trial (7.9km)
June 6: 1st stage: Aix-les-Bains to Givors (224km)
June 7: 2nd stage Givors to Chaufailles (187km)
June 8: 3rd stage: Roanne-Roanne, time trial (46.5km)
June 9: 4th stage: Tournon to Le Mont Ventoux (182km)
June 10: 5th stage: Vaison-la-Romaine to Grenoble (219km)
June 11: 6th stage: Albertville to Morzine-Avoriaz (155km)
June 12: 7th stage: Morzine-Avoriaz to Sallanches (115km)