The Step study of a recombinant adenovirus serotype 5 (Ad5)-based human immunodeficiency virus type 1 (HIV-1) vaccine revealed an increased risk of HIV-1 acquisition in vaccinees who were Ad5 seropositive at baseline. We therefore investigated whether preexisting Ad seropositivity to 7 different Ad serotypes was associated with increased risk of HIV-1 infection in 3 HIV-1 vaccine efficacy trials. In a case-control study involving 1570 adults enrolled in the VAX003 and VAX004 trials of a recombinant protein subunit HIV-1 vaccine and in the Step study, we observed that preexisting seropositivity to multiple Ad serotypes was not intrinsically associated with increased risk of HIV-1 acquisition.