A sensitive and specific polymerase chain reaction (PCR)-based assay was developed for four mutations in the reverse transcriptase gene of human immunodeficiency virus type 1 that have been associated with zidovudine resistance. These mutations were correlated in 366 specimens with zidovudine chemotherapy and resistance. Mutations at these four codons were detected only after zidovudine therapy. The usual sequence of appearance of mutations was codons 215, 70, 67, and 219, although individual variations occurred. The degree of resistance was proportional to the number of mutations present, although variable susceptibilities with identical patterns of mutations suggested the likelihood that additional mutations contribute to resistance. The existence of both phenotypic and genotypic mixtures was documented as was the occasional selection of subpopulations with passage of virus in vitro. The many complexities of zidovudine resistance render the assay of limited use for application to individual patients; however, it could prove useful for correlating disease or therapy with the emergence of resistance.