We have recently described a nonnucleoside compound that specifically inhibits the reverse transcriptase of human immunodeficiency virus type 1 (HIV-1), the causative agent of AIDS. This compound, nevirapine (BI-RG-587), interacts with highly conserved tyrosine residues at positions 181 and 188 in the reverse transcriptase to inhibit the recombinant enzyme and virus replication in cell culture with 50% inhibitory concentrations in the 40 nM range. HIV-1 variants resistant to nevirapine emerged with passage in cell culture in the presence of drug. This resistant phenotype was stable with continued passage in the absence of drug. These mutants had a substitution of cysteine for the tyrosine at position 181. Introduction of this mutation into the recombinant enzyme increased the inhibitory concentration of nevirapine 100-fold. Substitution of cysteine for tyrosine at residue 181 into the wild-type viral genome conferred a similar reduction in susceptibility to nevirapine. Mutants were also resistant to a tetrahydroimidazo[4,5,1-jk][1,4]benzodiazepin-2(1H)-one and -thione derivative and two 6-phenylthiouracil derivatives but retained their sensitivity to the other reverse transcriptase inhibitors, 3'-azido-3'-deoxythymidine and foscarnet.