The point mutation rate of a murine leukemia virus (MuLV) genome (AKV) was determined under conditions in which the number of replicative cycles was carefully controlled and the point mutation rate was determined by direct examination of the RNA genomes of progeny viruses. A clonal cell line infected at a low multiplicity of infection (2 x 10(-3)) was derived to provide a source of virus with high genetic homogeneity. Virus stocks from this cell line were used to infect cells at a low multiplicity of infection, and the cells were seeded soon after infection to obtain secondary clonal cell lines. RNase T1-oligonucleotide fingerprinting analyses of virion RNAs from 93 secondary lines revealed only 3 base changes in nearly 130,000 bases analyzed. To obtain an independent assessment of the mutation rate, we directly sequenced virion RNAs by using a series of DNA oligonucleotide primers distributed across the genome. RNA sequencing detected no mutations in over 21,000 bases analyzed. The combined fingerprinting and sequencing analyses yielded a mutation rate for infectious progeny viruses of one base change per 50,000 (2 x 10(-5)) bases per replication cycle. Our results suggest that over 80% of infectious progeny MuLVs may be replicated with complete fidelity and that only a low percentage undergo more than one point mutation during a replication cycle. Previous estimates of retroviral mutation rates suggest that the majority of infectious progeny viruses have undergone one or more point mutations. Recent studies of the mutation rates of marker genes in spleen necrosis virus-based vectors estimate a base substitution rate lower than estimates for infectious avian retroviruses and nearly identical to our determinations with AKV. The differences between mutation rates observed in studies of retroviruses may reflect the imposition of different selective conditions.