CTL activity is a major component of the host immune response associated with control of HIV replication in the course of infection. Emerging populations of HIV overcome the protective effector mechanisms with variant sequences unrecognized by CTL. Therefore, a critical element for containment of virus spread might be the establishment of an immune response against highly conserved epitopes. In this study, we selected a panel of nonamer or decamer peptides, with demonstrated binding affinity for HLA-A 0201, to define novel highly conserved envelope-derived epitopes of HIV-1. CTL activities were characterized from PBMC of five HLA-A2+, HIV-1-infected individuals given recombinant gp160. CTL activity derived from patient PBMC stimulated in vitro with peptide was demonstrated against at least two novel minimal env-encoded conserved epitopes. One epitope, KLTPLCVTL (aa 120-128), is highly conserved among HIV-1 strains of the B subtype. Analysis of a CTL clone reactivity to a distinct epitope (aa 814-823) demonstrated fluctuations in the recognition of peptides corresponding to natural virus variants found in vivo.