Most HIV-specific cytotoxic T-lymphocyte (CTL) epitopes have been identified using peptide-pulsed and recombinant vaccinia virus-infected targets. These systems may not accurately reflect the ability of epitopes to be presented by HIV-infected T cells. Recent studies suggest, in fact, that some CTL epitopes are poorly presented on HIV-infected cells. In this study, we have identified a novel A2.1-restricted HIV reverse-transcriptase (RT) epitope and investigated the presentation of this epitope by HIV-infected primary CD4+ T cells and T-cell lines. A CD8+ CTL clone, isolated from a seropositive subject that recognized a novel A2-restricted epitope KYTAFTIPSI (aa 293-302) in RT, was used for these studies. Primary CD4+ T cells and the CD4+ T-cell line T1 were infected with virus from T1-nPLAP, a cell line stably transfected with HXB-nPLAP, a molecular construct of HIV linked to a placental alkaline phosphatase (PLAP) marker gene. A uniformly infected cell population, obtained by immunomagnetic selection for PLAP expression, was used as targets in CTL assays. HIV-infected T cells were lysed by CTL recognizing this RT epitope as effectively as peptide-pulsed targets. This suggests that some RT epitopes are good targets for CTL recognition.