Treatment of HIV infection with zidovudine (ZDV) may select for changes in the genetic sequence of the viral reverse transcriptase (RT) that imparts drug resistance. The presence of a 2-bp mutation at codon 215 of RT (from threonine to phenylalanine or tyrosine) was assessed in plasma viral RNA in 85 subjects treated with ZDV in the AIDS Clinical Trials Group (ACTG) 175 virology substudy. Median CD4 cell numbers, HIV plasma RNA levels, and infectious titers of virus were significantly different over 56 weeks of treatment among 58 subjects with the wild-type threonine at codon 215 virus at study entry compared with the 27 subjects with mutations to phenylalanine or tyrosine (MUT) virus. Thirty percent (13 of 44 subjects) with wild-type virus at study entry developed a new codon 215 mutation. Genotypic resistance at codon 215 in plasma HIV RNA is associated with the subsequent immunologic and virologic failure of ZDV monotherapy in subjects with 200 to 500 CD4 cells/mm3.