Phelps, who has already qualified for the London Games in the 400 individual medley and 200 freestyle and is through to the 200 butterfly final later on Thursday, has never swum the 100 freestyle at the Olympics and only entered it as back up in the unlikely case he flopped in one of his other events.
Although he will not swim the individual 100 in London, the 26-year-old will still be involved in the 4x100 freestyle relay, just as he was in Athens in 2004 and Beijing in 2008.
Phelps's withdrawal from the 100 leaves him with a maximum of nine events for the Olympics although he is expected to drop the 200 backstroke and stick with the same eight events he competed in at the last two Games.
His rival Ryan Lochte did enter the 100 heats and qualified fifth fastest in the hope of making the 4x100 relay team for the first time.
Lochte, who has also qualified in the 400 medley and 200 freestyle, teamed up with Phelps to win gold medals in the 4x200 freestyle relay at the past two Olympics but has never swum in the shorter relay.
He won his 100 heat on Thursday in 49.33 seconds, storming home after turning in seventh place.
Scot Robertson set the fastest time (49.08) ahead of Adrian Nathan (49.17) and Matt Grevers (49.24), with the top 16 advancing to the semi-finals.
Jason Lezak, the 36-year-old whose stunning anchor leg swim in Beijing set the platform for Phelps's record eighth gold medal, was ninth overall in 49.40 and needs to finish in the top eight from the semi-finals to make the final.
Clark Burckle (2:10.30) set the fastest time in the men's 200 breaststroke heats while cancer survivor Eric Shanteau (2:11.57) and 100 breaststroke winner Brendan Hansen (2:12.14) were third and fifth respectively.
Kim Vandenberg (2:08.78) led the way in the women's 200 butterfly preliminaries.
There are three finals scheduled on Thursday night - the men's 200 butterfly, women's 200 freestyle and women's 200 medley.