A comparison of the fidelity of reverse transcriptases (RT) from human immunodeficiency virus (HIV-1) and avian myeloblastosis virus (AMV) is made using RNA and DNA primer-template molecules in vitro. Selected template target sites containing either uracil or thymine are used to measure nucleotide insertion fidelities and to compare the efficiency of extending mismatched nucleotides at primer 3'-termini. HIV-1 reverse transcriptase is observed to incorporate as many as three consecutive mismatches and to continue efficient elongation from mismatched primer 3'-termini without discernible pausing. Nucleotide misinsertion and mispair extension efficiencies are similar for both enzymes on RNA and DNA templates having identical surrounding sequence. HIV-1 and AMV reverse transcriptases form G.T and G.U mismatches most efficiently, between 1.6 x 10(-4) and 7 x 10(-4), and both enzymes extend G.U with exceptionally high efficiencies, 2.7 x 10(-2) for HIV-1 RT and 4.5 x 10(-2) for AMV RT. Extension of the G.T mismatch is similar for AMV RT (5.8 x 10(-2) but 20-fold less efficient for HIV-1 RT. C.U and C.T mismatches are formed by both enzymes in a frequency range of 4.4 x 10(-5)-2.4 x 10(-4). HIV-1 RT extends these mismatches with slightly higher efficiencies (5.5 x 10(-3)-5.9 x 10(-3)) than AMV RT (5.6 x 10(-4)-2.1 x 10(-3)). Insertion of dTMP opposite U and T occur at about 1 x 10(-4)-2 x 10(-4) for HIV-1 RT. For AMV RT, formation of T.U mispairs occurs with an 8-fold lower efficiency, whereas insertion of dTMP opposite T is not detected. This particular DNA template sequence generates a pause site for AMV RT but not HIV-1 RT. HIV-1 RT dissociation rate constants are about 8-fold larger from a DNA primer bound to a DNA template (0.5 s-1), as compared with an RNA template (0.06 s-1) at one site, and are at most 2-fold larger at another site. The equilibrium binding constant for HIV-1 RT bound to DNA primed RNA and DNA templates appears to be similar, KD approximately 2.5 nM. Values of kpol from 0.3 to 1.5 nucleotides/s are obtained for HIV-1 RT at the RNA and DNA template sites used to measure insertion and extension fidelity. The relatively high efficiency of mispair extension catalyzed by reverse transcriptases with both RNA and DNA templates suggests that a significant component of retroviral genetic variability may be related to the ability of reverse transcriptases to continue efficient synthesis of DNA containing mismatches on both RNA and DNA templates.