The rejection of allografts in mammals is mainly mediated by cytotoxic T-lymphocytes, whereas no comparable immunoreactive cells have been described in invertebrates. The present study was undertaken to determine whether similar cytotoxic effector cells are present when allograft rejection occurs in the terrestrial slug Incilaria fruhstorferi. A piece of dorsal skin from a donor animal was orthotopically transplanted to a recipient. Immunohistochemistry for perforin, detection of apoptosis by the TUNEL (TdT-mediated dUTP-biotin nick-end labeling) method, and electron microscopy were performed using both donor and recipient tissues. Cellular changes in the rejection process continued over for 40 days. Two functional types of "effector" cells were recognized at the rejection site, but they were observed to be macrophages possessing perforin granules and phagocytosing damaged cells of the allograft. Three days after transplantation, the perforin-positive cells were recognized only in the recipient tissue surrounding the allograft. Five days after transplantation, these cells started to appear in the graft, while they disappeared from the host tissue. However, TUNEL-positive cells were not observed throughout the graft-rejection process. Electron microscopic examination of the graft tissue revealed autophagic degeneration of epithelial cells, mucous cells, pigment cells, fibroblasts, and muscle cells. These observations suggest that the molluscan slug has the capability to recognize differences in cell-surface molecules between the allogeneic and recipient tissues, and that an allograft is chronically rejected due to a type of immunocyte that can induce perforin-dependent cell death.