Researchers at the National Cancer Institute first recognized the anti-HIV potential of the carboxanilide compound oxathiin carboxanilide (UC84) [Bader, J. P., et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 6740-6744]. We have compared the inhibitory effect of UC84 and a second-generation thiocarboxanilide derivative, UC38, on HIV-1 reverse transcriptase (RT) RNA-dependent DNA polymerase activity. UC38 was a much better inhibitor (IC50 = 0.8 microM) than UC84 (IC50 = 4.3 microM). Inhibition by UC84 was competitive with respect to primer/template (P/T), whereas that by UC38 was uncompetitive. Both compounds were mixed noncompetitive inhibitors with respect to deoxynucleoside triphosphate (dNTP). Both compounds protected RT from irreversible photoinactivation by an azido derivative of nevirapine, implying that UC84 and UC38 bind to the same region of RT as nevirapine. UC84 photoprotected both free RT and the RT-P/T binary complex, but did not protect the RT-P/T-dNTP ternary complex. In contrast, UC38 completely photoprotected the RT-P/T-dNTP ternary complex, but not free RT or the RT-P/T binary complex. UC84 and UC38 thus appear to bind to different mechanistic forms of RT in the polymerase reaction sequence.