A system has been designed to study the in vivo forward rate of mutation of human immunodeficiency virus type 1 (HIV-1) during one round of replication. A HIV-1 shuttle vector was used that contained the lacZα peptide gene as a reporter for mutations. The forward mutation rate of HIV-1 was found to be 3 x 10-5 mutations per target base pair per cycle, or about 20-fold lower than the error rates reported for purified HIV-1 reverse transcriptase with sense-strand RNA and DNA templates of the lacZα peptide gene in a cell-free system. To test the hypothesis that the vpr gene product might, at least in part, account for the lower mutation rate observed in vivo, a HIV-1 vector was replicated to determine if the mutation rate was higher in the absence of the wild-type vpr gene product. A vpr- shuttle vector had an overall mutation rate as much as 4-fold higher than that of the parental vector. A shuttle vector with an amino acid substitution in Vpr that prevents efficient incorporation of Vpr into virus particles was found to have a mutation frequency similar to that of the vpr- vector, and was interpreted to indicate a requirement for Vpr incorporation into the virus particle in order to observe the influence of vpr on the mutation rate. Replication of a vpr- shuttle vector in the presence of a wild-type vpr expression plasmid led to a mutation frequency similar to that of the parental vector, suggesting that the vpr mutation could be complemented in trans. Immunoprecipitation analysis indicated that Vpr virion incorporation coincided with the influence of vpr on the mutation rate.