Variation in gene sequences from human immunodeficiency virus type 1 (HIV-1) p17gag and the third hypervariable region (V3 loop) of env was examined in a cross-sectional study of single specimens from 4 HIV-1 polymerase chain reaction-positive, seronegative injection drug users. As observed in sexually transmitted disease, clones of HIV-1 genes isolated from an individual were remarkably homogeneous, with amino acid variation in the envelope region of 0.3%-0.9% and in p17gag of 0.0%-0.7%. Intersubject variation was much greater (at the amino acid level, 15% for the envelope and 4.3% for p17gag), with conservation in the envelope only of regions at the base of the V3 loop. Since at least some subjects likely acquired infection intravenously, the ability of virus to survive in seminal fluid or on mucosal surfaces cannot explain the observed homogeneity, which may depend on direct interactions between the virus and the infected cell.