Accumulating data have brought the nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) into the forefront of antiretroviral therapy. Among the emerging compounds in this class, a particularly attractive one is efavirenz (Sustiva), recently approved for clinical use by the U.S. Food and Drug Administration. In the present study, the equilibrium dissociation constants for efavirenz binding to the different catalytic forms of human immunodeficiency virus type 1 RT as well as the association and dissociation rates have been determined using a steady-state kinetic approach. In addition, the same enzymological analysis has been extended to the thio-substituted analog, sefavirenz, which showed comparable activity in vitro against RT. Both compounds have been found to act as purely uncompetitive inhibitors at low drug concentrations (5 to 50 nM) and as mixed noncompetitive inhibitors at higher doses (50 to 500 nM). This behavior can be interpreted in terms of the relative affinities for the different catalytic forms of the enzyme. Both efavirenz and sefavirenz showed increasing affinities for the different forms of RT in the following order: free enzyme < (i.e., bound with lower affinity) binary RT-template-primer (TP) complex < ternary RT-TP-deoxynucleoside triphosphate (dNTP) complex. The rate of binding of the two inhibitors to the different enzyme-substrate complexes was well below the diffusion limit (on the order of 10(4) M(-1) s(-1)); however, both inhibitors, when bound to the ternary RT-TP-dNTP complex, showed very low dissociation rates, on the order of 10(-4) s(-1) for both compounds, typical of tightly binding inhibitors. Thus, efavirenz and its thio-substituted derivative sefavirenz appear to be peculiar in their mechanism of action, being selective tightly binding inhibitors of the ternary RT-TP-dNTP complex. Efavirenz is the first clinically approved NNRTI to show this property.