It has been more difficult to prevent intestinal allograft rejection than rejection of other solid organ transplants. Epithelial cells in the small intestine have the potential to actively participate in graft rejection because they constitutively express MHC class II antigens. The present study examined the ability of intestinal epithelial cells (IEC) to stimulate allogeneic T cells in vitro using one-way MLC. IEC from jejunum and ileum of ACI rats (RT1a) were used as the stimulator cells; purified T lymphocytes from Lewis rats (RT11) were used as the responder cells. Freshly isolated IEC were poor stimulators of naive allogeneic T lymphocytes. However, IEC induced a strong proliferation of primed allogeneic T lymphocytes, and this response could be blocked by anti-MHC class II mAb (OX-4). IEC stimulated a vigorous proliferation of naive T cells when co-cultured with (1) macrophages syngeneic with responder T lymphocytes, (2) supernatants of LPS-stimulated macrophages, or (3) a combination of IL-1 and IL-6. We conclude that MHC class II positive IEC require soluble costimulatory factors to fully activate naive allogeneic T cells. Immune stimulation by enterocytes may be one of the reasons why it is so difficult to prevent intestinal allograft rejection. Strategies that block interactions among IEC, macrophages, and lymphocytes may reduce the immunogenicity of small bowel transplants.