The evolution of HIV-1 sequences over time is the result of the selection of mutant variants that have escaped from host immune responses or the outgrowth of mutants with increased viral replication, or both. We investigated the contribution of both selection processes to the overall evolution of the Tat and Rev regulatory gene sequences from four individuals, ranging in time from just prior to seroconversion to stable asymptomatic infection. After sequencing at least 15 clones per sample per gene, we analyzed the sequence evolution of the MHC-I motifs that were predicted from the MHC-I haplotypes of these patients. For each identified Tat sequence, we tested the activity of the corresponding encoded protein in a transactivation assay in vitro. Our results suggest that the evolution of the Tat and Rev sequences from these individuals can be explained by mutational escape of the MHC-I epitopes and that no mutations that replaced the original sequences in the viral population are associated with either an increase or decrease in Tat activity. CTL-mediated selection appears to be an important determinant of HIV-1 regulatory gene sequence evolution during the early stages of infection.