We have generated transgenic mice by introducing into fertilized eggs the major histocompatibility complex class I gene, H-2Kb, linked to various promoters that target gene expression to particular tissues. In one system the gene was expressed in the medullary epithelial cells of the thymus; these were unable to impose tolerance on differentiating T lymphocytes. These findings, together with those of other workers who showed that epithelial thymus grafts can impose tolerance, provide clear evidence of functional heterogeneity among thymus epithelial cells. In other transgenic models the gene was expressed in nonlymphoid tissues, such as the pancreatic islet beta cells or the hepatocytes, exocrine pancreas and kidney tubules. In all these cases the tissues were not subjected to autoimmune attack and the animals were specifically tolerant of H-2Kb-bearing cells in vivo, although some could generate cytotoxic T lymphocytes in vitro. Hence, although the intrathymic environment may be the dominant site for negative selection, it is clear that ancillary mechanisms exist in the periphery to ensure that tolerance is achieved to antigens not synthesized in the thymus.