The role of Moloney murine leukemia virus (MoMLV) reverse transcriptase (RT) in the generation of base substitution mutations during retroviral replication was analyzed. To that effect, the in vitro fidelity of the MoMLV RT was compared to the rate of base substitution mutations occurring during the replication of an MoMLV-based retroviral vector. Using the vector in an amber reversion assay, the base substitution mutation rate at a single locus was found to be 2 x 10(-6)/base pair in one cycle of vector virus replication. Analysis of the fidelity of the purified RT using the same template sequence revealed that, of the two mispairs (A.C and T.G) that would lead to reversion of the amber codon during replication, A.C occurs at a rate of 4.0 x 10(-6), and T.G occurs at a rate of 0.7 x 10(-4). While the rate of formation of A.C is very similar to the vector mutation rate, the rate of formation of T.G is more than 30 times higher. This discrepancy in rates suggests that there are other elements in the infected cells that contribute to the fidelity of viral replication.