Variants of human immunodeficiency virus (HIV) with reduced sensitivity to zidovudine (3'-azido-3'-deoxythymidine) have been selected by passage of virus in cell culture in the presence of drug. Wild-type, sensitive virus became partially resistant to zidovudine by passage 12 (50% inhibitory dose values measured in HeLa CD4+ cells increased from 0.014 to 0.2 microM), and genetic analysis using the polymerase chain reaction revealed that mutations in the reverse transcriptase coding region identical to those seen in clinical isolates from treated individuals had occurred. The order of appearance of these resistance mutations in passaged virus was also similar to that in clinical isolates. The partially resistant strain, HIVRTMC/F, became highly zidovudine resistant by passage 12 (50% inhibitory dose values increased from 0.4 to 2.5 microM during passages 7 to 11). Nucleotide sequence analysis of the reverse transcriptase from this variant revealed a novel amino acid substitution (Lys----Glu) at codon 219. A different substitution at this codon (Lys----Gln) had been seen previously in clinical isolates. When this mutation was created in HIVRTMC/F by site-directed mutagenesis, the resulting partially resistant virus became highly resistant, thus confirming the significance of this change. In view of the possibility that this mutation might occur in HIV isolates during treatment of patients, we adapted our selective polymerase chain reaction procedure to enable screening for this change in clinical samples. The virus passage procedure described here may be useful for gaining further insight into the mutational events occurring during the development of resistance to zidovudine and other HIV inhibitors.