We compared the mechanistic and kinetic properties of murine leukemia virus (MuLV) and human immunodeficiency virus type 1 (HIV-1) reverse transcriptases (RTs) during RNA-dependent DNA polymerization and mutation synthesis using pre-steady-state kinetic analysis. First, MuLV RT showed 6.5-121.6-fold lower binding affinity (K(d)) to deoxynucleotide triphosphate (dNTP) substrates than HIV-1 RT, although the two RTs have similar incorporation rates (k(pol)). Second, compared with HIV-1 RT, MuLV RT showed dramatic reduction during multiple dNTP incorporations at low dNTP concentrations. Presumably, due to its low dNTP binding affinity, the dNTP binding step becomes rate-limiting in the multiple rounds of the dNTP incorporation by MuLV RT, especially at low dNTP concentrations. Third, similar fold differences between MuLV and HIV-1 RTs in the K(d) and k(pol) values to correct and incorrect dNTPs were observed. This indicates that these two RT proteins have similar misinsertion fidelities. Fourth, these two RT proteins have different mechanistic capabilities regarding mismatch extension. MuLV RT has a 3.1-fold lower mismatch extension fidelity, compared with HIV-1 RT. Finally, MuLV RT has a 3.8-fold lower binding affinity to mismatched template/primer (T/P) substrate compared with HIV-1 RT. Our data suggest that the active site of MuLV RT has an intrinsically low dNTP binding affinity, compared with HIV-1 RT. In addition, instead of the misinsertion step, the mismatch extension step, which varies between MuLV and HIV-1 RTs, contributes to their fidelity differences. The implications of these kinetic differences between MuLV and HIV-1 RTs on viral cell type specificity and mutagenesis are discussed.