Pyridinone derivatives are potent and specific inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and HIV-1 replication in cell culture. However, the potential clinical usefulness of these compounds as monotherapeutic agents may be limited by the selection of inhibitor-resistant viral variants. Resistance in cell culture is due primarily to mutational alterations at RT amino acid residues 103 and 181. A recombinant HIV-1 RT containing both of these mutations was used to screen a panel of pyridinone analogs for inhibitory activity. L-696,229 and L-697,661, pyridinones currently undergoing clinical evaluation, were more than 4,000-fold weaker against the mutant enzyme than against the wild-type enzyme. In contrast, one derivative of L-696,229, L-702,019 (3-[2-(4,7-dichlorobenzoxazol-2-yl)ethyl]-5-ethyl-6-methylpyrid in-2(1H)-thione), showed only three-fold different potencies against the two enzymes. L-702,019 was also a potent inhibitor of the replication of mutant HIV-1 containing the individual mutations at amino acid 103 or 181 as well as of clinical isolates resistant to L-697,661 and L-696,229. Isolation and analysis of resistant viral variants in cell culture showed that significant resistance to L-702,019 could be engendered only by multiple amino acid substitutions in RT. Accordingly, these studies demonstrated the potential of identifying second-generation specific HIV-1 RT inhibitors that can overcome the viral resistance selected by the first generation of inhibitors.