CTL directed at the highly conserved HIV-1 gag protein have been described in HIV-1 seropositive persons and may be an important host defense against this retrovirus. Presently only limited data are available regarding the specific epitopes recognized by these CTL. In this study, we have performed a detailed examination of the gag-specific CTL response in three HIV-1 seropositive subjects, using both unstimulated PBMC and cloned CTL. Lysis of gag-expressing targets was found to be mediated by CD3+CD8+ lymphocytes and restricted by class I Ag. Multiple class I Ag were found to restrict gag epitopes in each subject studied, with as many as three of these Ag involved in presenting gag CTL epitopes in a single subject. The majority of gag-specific CTL activity was found to be directed against epitopes in the p24 subunit of the gag protein, with at least seven different HLA class I-restricted CTL p24 epitopes identified in these three subjects. Less CTL activity was directed against p17 subunit of gag and two CTL epitopes were identified in this protein. Although as many as four different epitopes in gag were recognized using CTL from a single subject, none of the epitopes was recognized by CTL from more than one subject. Analysis of gag epitope recognition using cloned CTL demonstrated heterogeneity and specificity not appreciated using unstimulated PBMC. The identification of multiple relatively conserved epitopes in the HIV-1 gag protein and the heterogeneity of CTL responses to this protein may have important implications for vaccine development and our understanding of AIDS pathogenesis.