Virus isolates resistant to 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) and a highly potent HEPT derivative, [1-benzyloxymethyl-5-ethyl-6-(alpha-pyridylthio)uracil] (NSC 648400, E-BPTU), were selected in cell culture. Cross-resistance evaluation indicated that the two drug-resistant virus isolates were phenotypically distinct from one another although each of the virus isolates was resistant to both of the HEPT derivatives. The virus isolate resistant to NSC 648400 had a single amino acid change in the reverse transcriptase (Y181C) which resulted in cross-resistance to all of the nonnucleoside reverse transcriptase inhibitors evaluated, with the exception of calanolide A. The NSC 648400-resistant virus isolate exhibited 15-fold enhanced sensitivity to calanolide A. The virus isolate selected in the presence of HEPT exhibited a single amino acid change (P236L) which was not cross-resistant to other nonnucleoside RT inhibitors tested with the exception of the two HEPT derivatives. This HEPT-resistant virus isolate exhibited enhanced sensitivity (5- to 10-fold) to thiazolobenzimidazole. We have used both virus isolates with defined single amino acid changes in the RT and bacterially expressed RTs with site-directed amino acid substitutions to test the effects of a wide variety of mutations on the activity of NSC 648400. Single mutations at amino acids 101, 103, 106, 181, or 236 yielded virus with high resistance (> 20-fold) to NSC 648400, while lower levels of resistance were seen with mutations at amino acids 98, 100, or 108. These results suggest that several changes in the conformation of the nonnucleoside inhibitor binding site of the HIV-1 reverse transcriptase can affect the inhibitory activity of the HEPT class of compounds.