To decipher the mechanism for non-nucleoside inhibitor resistance of HIV-1 reverse transcriptase, the kinetics of the interaction between wild type and drug-resistant variants of the enzyme and structurally diverse inhibitors were determined. Substitution of amino acid residues in the inhibitor binding site resulted in altered rate constants for the pre-equilibrium between two unliganded forms of the enzyme, and for the association and dissociation of the inhibitor-enzyme interaction. The Y181C, V108I, and P225H substitutions affected primarily the association and dissociation rate constants, while the K103N and the L100I substitutions also influenced the equilibrium between the two forms of the free enzyme. The K103N and the L100I substitutions were found to facilitate both the entry of the inhibitor into the binding pocket as well as its exit, in contrast to what has been reported elsewhere. Interaction kinetic-based resistance profiles showed that phenethylthiazolylthiourea compounds were relatively insensitive to the studied substitutions.