A common target for therapies against human immuno-deficiency virus type 1 (HIV-1) is the viral reverse transcriptase (RT). Treatment with the widely used nucleoside analog (-)-2', 3'-deoxy-3'-thiacytidine (3TC) leads to the development of resistance-conferring mutations at residue M184 within the YMDD motif of RT. First, variants of HIV with the M184I substitution appear transiently, followed by viruses containing the M184V substitution, which persist and become the dominant variant for the duration of therapy. In the three-dimensional crystal structure of HIV-1 RT complexed with double-stranded DNA, the M184 residue lies in the vicinity of the primer terminus, near the incoming dNTP substrate. Recent studies have shown that 3TC resistance mutations, including M184I, increase the nucleotide insertion and mispair extension fidelity. Therefore, we have examined the effects of the M184I mutation on the overall polymerase fidelity of HIV-1 RT via an M13-based forward mutation assay. We found the overall error rate of the M184I variant of HIV-1 RT to be 1.7 x 10(-5) per nucleotide. This represents a 4-fold increase in fidelity over wild-type HIV-1Hxb2RT (7.0 x 10(-5) per nucleotide) and a 2.5-fold increase in fidelity over the M184V variant (4.3 x 10(-5) per nucleotide). Of the nucleoside analog resistance mutations studied using the forward assay, the M184I variant has shown the greatest increase in fidelity observed to date. Interestingly, the M184I variant RT displays significantly altered error specificity, both in terms of error rate at specific sites and in the overall ratio of substitution to frameshift mutations in the entire target.