Background. Porcine endogenous retroviruses (PERV) have been shown to infect human cells, raising concerns regarding safety of xenotransplantation. In patients exposed to porcine tissues, no PERV infection has been observed. This study was designed to develop human CD8+ cytotoxic T lymphocytes (CTL) against PERV-derived peptides presented in the context of human leukocyte antigen (HLA) or swine leukocyte antigen (SLA) class I molecules and to define dominant epitopes contributed by PERV. Methods. Human CD8+ CTL were generated against porcine aortic endothelial cells (PAEC). Peptides presented on SLA class I molecules were acid eluted and fractionated by reverse-phase high-performance liquid chromatography. Peptide fractions that restored lysis of acid-stripped PAEC were sequenced by tandem mass spectrometry. Human CD8+ CTL were generated against PERV envelope-derived peptides and PERV-infected human cells to identify immunodominant PERV-derived epitopes. Results. We identified two peptides derived from retroviral transactivating regulatory protein (AHQDPLPEQP) and retroviral transcription factor (PQKPFVT) recognized by human CD8+ CTL in the context of SLA class I. Computer-assisted analysis identified nine PERV-envelope-derived 9-mer peptides with high affinity for the HLA-A2 molecule (Env-1-9). PERV-specific CD8+ CTL generated in vitro identified the immunodominant Env-5 peptide (303-311, KLFSLIQGA) and demonstrated HLA-A2-restricted cytotoxicity against PERV-infected human cells. Conclusions. Our results indicate that PERV-derived peptides are presented naturally on porcine and human major histocompatibility complex class I molecules. CD8 + CTL responses elicited against dominant SLA and HLA class I-restricted PERV-derived epitopes may play an important role in xenograft rejection and in containment of PERV infection of human cells after xenotransplantation.