Presence of mutations associated with resistance to zidovudine or lamivudine was determined in isolates of HIV-1 obtained after long-term follow-up of 64 infected individuals who received zidovudine, lamivudine, or both drugs as initial antiretroviral therapy. Zidovudine resistance mutations were less frequent in isolates from patients treated with combination lamivudine plus zidovudine compared with zidovudine alone, but these mutations accumulated over time. Phenotypic resistance to both drugs was found in isolates from 3 of 23 patients. In 3 other patients, lamivudine-resistant virus detected at week 12 was replaced by wild-type virus after longer follow-up, which correlated with a return to baseline levels of plasma HIV-1 RNA. These results show that dual resistance to zidovudine and lamivudine develops over time despite the delayed emergence of zidovudine-resistant mutations. These results also suggest a selective advantage in vivo for HIV-1 species that are wild-type at RT codon 184.