Recently, several classes of compounds have been shown to be extremely selective inhibitors of human immunodeficiency virus type 1 (HIV-1) replication in vitro. These include the tetrahydro-imidazo[4,5,1-jk][1,4]-benzodiazepin-2(1H)-one and -thione (TIBO), 1-(2-hydroxyethoxymethyl)-6-(phenylthio)-thymine (HEPT), dipyridodiazepinone, pyridinone and bis(heteroaryl)piperazine derivatives. The hallmark of these new antiviral compounds is a specific interaction with reverse transcriptase (RT) of HIV-1. They are inactive against HIV-2 and any other viruses tested. Here we describe that, in addition to the HIV-1 strains, two simian immunodeficiency virus (SIV) strains from African green monkeys (SIVagm3 and SIVagmTYO-1) are also sensitive to the TIBO class of compounds. TIBO and HEPT derivatives block the replication of SIVagm in cell culture at micromolar concentrations. Kinetics of inhibition of SIVagm RT by TIBO are competitive with respect to the natural substrate (dGTP). Amino acid alignments and site-directed mutagenesis point to the critical role of amino acid residues Y181 and Y188 in the sensitivity of HIV-1 RT and SIVagm RT to inhibition by the TIBO derivatives. Antiviral efficacy studies with this range of compounds and using sensitive SIV strains are now feasible in monkeys.