Abstract: Miniature swine have a variety of advantages as potential donors for human xenotransplantation, including size, physiological similarities, and breeding characteristics. To investigate the nature of the human anti‐pig xenogeneic cellular response, we performed standard 51Cr‐release cell‐mediated lympholysis (CML) experiments. The major histocompatibility complex (MHC) allele specificity of the xenogeneic cellular response was tested on porcine target cells of three distinct homozygous MHC haplotypes (SLAaa, SLACC, SLAdd) and three intra‐MHC recombinant haplotypes (SLAjj, SLAgg, SLAkk), obtained from our herd of partially inbred miniature swine. After stimulation with irradiated porcine peripheral blood mononuclear cells (PBMC) of SLAaa haplotype, a strong nonspecific cytotoxic response of the bulk culture against xenogeneic targets of all three haplotypes was observed. However, SLA allele specificity could be demonstrated after T cell enrichment, and mapping experiments revealed predominantly SLA class I restriction of xenoreactive cytotoxic T lymphocytes (CTLs), although some class II restriction was also observed. The experiments were repeated in the presence of anti‐T cell monoclonal antibodies, anti‐CD3 (OKT3), anti‐CD2 (35.1), anti‐CD4 (OKT4), or anti‐CD 8 (OKT8). The bulk xenogeneic CML was not inhibited by any of the anti‐T cell antibodies tested. However, after T cell‐enrichment, lysis of porcine targets was significantly inhibited by anti‐CD3 or anti‐CD8 antibody and partially inhibited by anti‐CD2 antibody. In comparable assays, the human allogeneic CML was blocked by anti‐CD3 and anti‐CD8, but not by anti‐CD2 or anti‐CD4 antibodies. Finally, the cytotoxic activity of A3b3, a human CD4+ T‐cell clone, was tested. A3b3 lysed xenogeneic targets of SLAaa haplotype, but not SLACC or allogeneic targets, and was inhibited by anti‐CD4, anti‐CD2, and anti‐CD3 antibodies, but not by anti‐CD8. With the aid of intra‐MHC recombinant haplotypes, the xenogeneic CML reactivity of A3b3 was mapped to SLA class II, suggesting direct xenogeneic recognition of porcine MHC class II antigens by human T cells. Thus, the human anti‐pig cell‐mediated cytotoxic response is similar in magnitude to comparable allogeneic responses, and involves both SLA class I and class II restricted T‐cell mediated cytotoxicity, as well as additional nonspecific killing, possibly by NK cells.