A single amino acid substitution from methionine-184 to valine (M184V) of HIV-1 reverse transcriptase (RT) evokes the 1000-fold 3TC (Lamivudine) resistance by the HIV-1 virus observed in the clinic. The M184V mutant HIV-1 RT was studied to assess its catalytic efficiency during single nucleotide incorporation using a transient kinetic approach. The maximum rate of polymerization (k(pol)), binding affinity (K(d)), and incorporation efficiency (k(pol)/K(d)) were determined for incorporating dCTP and 3TC-TP by wild-type and 3TC-resistant HIV-1 RT. The 3TC-resistant HIV-1 RT showed a similar efficiency of incorporation compared with the wild-type enzyme during DNA-dependent DNA polymerization; however, the incorporation efficiency is reduced 3.5-fold during RNA-dependent polymerization. A dramatic 146- and 117-fold decrease in incorporation efficiency was observed for 3TC-MP incorporation by M184V RT for DNA- and RNA-dependent DNA polymerization, respectively, as compared with wild-type HIV-1 RT. While the k(pol) was slower and the K(d) was weaker for 3TC-TP incorporation by the M184V RT, the decrease in the efficiency of incorporation is primarily due to a substantially reduced binding affinity for the 3TC-TP to the enzyme.DNA (or RNA) complex poised for DNA elongation. The fidelity of M184V RT was also examined to evaluate mispair formation since this mutant has been suggested to exhibit a higher level of fidelity. The results of our studies indicate that there is a maximum 2.4-fold increase in fidelity for M184V RT as compared with wild-type HIV-1 RT. Both the wild-type and 3TC-resistant mutant RT showed higher fidelity using an RNA template as contrasted with the corresponding DNA template. This mechanistic information provides insight into our understanding of the molecular mechanism of 3TC-drug resistance and supports suggestions that increased RT fidelity and decreased fitness of the M184V HIV-1 virus may be factors contributing to the strong antiviral effect of AZT-3TC combination therapy.