Neonatally thymectomized (NTx) mice fall victim to such autoimmune diseases as gastritis and pancreatitis with aging. Self-reactive T cell clones are known to be consistently generated through TCR intermediate (i.e. TCRint) cell differentiation in normal mice (i.e. via the extrathymic pathways and an alternative intrathymic pathway). It was investigated whether the generation of such clones in NTx mice follows this rule or whether they are generated by default via mainstream T cell differentiation in the thymus. The majority of T cells generated in NTx mice were TCRint cells in all organs tested. In contrast to athymic mice, which carry only TCRint cells with aging, a leaky appearance of high TCR (i.e. TCRhi) cells emerged in the lymph nodes and other organs of NTx mice. Self-reactive clones estimated by anti-Vbeta monoclonal antibodies in conjunction with the Mls system were confined to TCRint cells in all tested organs, including a target organ, the stomach, in NTx mice. A leaky population of TCRhi cells did not contain a significant number of self-reactive clones. Moreover, such self-reactive clones among TCRint cells in NTx mice with autoimmune disease were shown to be nonanergic in the proliferation assay. The present results suggest that the generation of self-reactive clones is totally due to TCRint cell differentiation, although it is still undetermined whether the expanding TCRint cell population is generated via the extrathymic pathway or an alternative intrathymic pathway. It is shown here not to be due to a failure of the TCRhi cell-differentiation pathway in NTx mice.