Variants of human immunodeficiency virus type 1 (HIV-1) that are highly resistant to a number of nucleoside analog drugs have been shown to develop in some patients receiving 2',3'-dideoxy-3'-azidothymidine therapy in combination with 2',3'-dideoxycytidine or 2',3'-dideoxyinosine. The appearance, in the reverse transcriptase (RT), of the Q151M mutation in such variants precedes the sequential appearance of three or four additional mutations, resulting in a highly resistant virus. Three of the affected residues are proposed to lie in the vicinity of the template-primer in the three-dimensional structure of the HIV-1 RT-double-stranded DNA complex. The amino acid residue Q151 is thought to be very near the templating base. The nucleoside analog resistance mutations in the beta9-beta10 (M184V) and the beta5a (E89G) strands of HIV-1 RT were previously shown to increase the fidelity of deoxynucleoside triphosphate insertion. Therefore, we have examined wild-type HIV-1BH10 RT and two nucleoside analog-resistant variants, the Q151M and A62V/V75I/F77L/F116Y/Q151M (VILYM) RTs, for their overall forward mutation rates in an M13 gapped-duplex assay that utilizes lacZ alpha as a reporter. The overall error rates for the wild-type, the Q151M, and the VILYM RTs were 4.5 x 10(-5), 4.0 x 10(-5), and 2.3 x 10(-5) per nucleotide, respectively. Although the mutant RTs displayed minimal decreases in the overall error rates compared to wild-type RT, the error specificities of both mutant RTs were altered. The Q151M RT mutant generated new hot spots, which were not observed for wild-type HIV-1 RT previously. The VILYM RT showed a marked reduction in error rate at two of the predominant mutational hot spots that have been observed for wild-type HIV-1 RT.