The mechanism of inhibition of HIV-1 reverse transcriptase by three nonnucleoside inhibitors is described. Nevirapine, O-TIBO, and Cl-TIBO each bind to a hydrophobic pocket in the enzyme-DNA complex close to the active site catalytic residues. Pre-steady-state kinetic analysis was used to establish the mechanism of inhibition by these noncompetitive inhibitors. Analysis of the pre-steady-state burst of DNA polymerization indicated that inhibitors blocked the chemical reaction, but did not interfere with nucleotide binding or the nucleotide-induced conformational change. Rather, in the presence of saturating concentrations of the inhibitors, the nucleoside triphosphate bound tightly (K-d, 100 nM), but nonproductively. The data suggest that an inhibitor combining the functionalities of a nonnucleoside inhibitor and a nucleotide analog could bind very lightly and specifically to reverse transcriptase and could be effective in the treatment of AIDS.