The dipyridodiazepinone human immunodeficiency virus type 1 (HIV-1)-specific reverse transcriptase (RT) inhibitor BI-RG-587 was tested for its ability to inhibit HIV-1 replication in both acutely and chronically infected cell lines. The ability of BI-RG-587 to inhibit steps in the virus replicative cycle other than reverse transcription was also assessed. BI-RG-587 was found to be a potent inhibitor of HIV-1 replication in acutely infected cells (50% inhibitory concentration [IC50] = 37.2 nM), and the sensitivity and kinetics of that inhibition was similar to the known RT inhibitor zidovudine (AZT). Even at 100x IC50, BI-RG-587 had no effect on gp120/CD4 interaction, syncytia formation, or envelope glycoprotein processing. In addition, no inhibition of viral replication or protein production was noted in a chronically infected cell line that produces viral products in an RT-independent manner. Finally, no inhibition of acute HIV-2 replication was noted, even with very high (2500x IC50 for HIV-1) concentrations of BI-RG-587. These results demonstrate that BI-RG-587 is a potent inhibitor of HIV-1 replication and that this inhibition occurs at the point of reverse transcription.