Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) variants with the K65R or L74V substitution display resistance to several nucleoside analogs. An in vitro dNTP exclusion assay revealed an increased fidelity for K65R RT compared with wild-type RT, but little change for L74V RT. When the forward mutation rates were measured via a gap-filling assay, the K65R variant displayed an 8-fold decrease in the overall mutation rate (1.0 x 10(-3) versus 8.6 x 10(-3) for wild-type HIV-1 RT), whereas the rate for the L74V variant was closer to that for wild-type RT (5.0 x 10(-3)). The increase in overall fidelity observed for K65R RT is the largest reported for any drug-resistant HIV-1 RT variant. Nucleotide sequence analysis of lacZalpha mutants generated by variant RTs indicated that K65R RT displays uniform reduction in most types of errors, whereas L74V RT does not. Modeling the substitutions into the x-ray structure of the ternary complex revealed that the major influence of Leu(74) in stabilizing the templating base is unaffected by Val substitution, whereas the K65R substitution appears to increase the stringency of dNTP binding. It is speculated that the increased fidelity of K65R RT is due to an altered interaction with the dNTP substrate.