Congenitally athymic nude (nu/nu) mice have been thought to exhibit no T cell functions despite the presence of some Thy-1 positive (Thy-1+) cells. However, we detected a significant number of Con A-responsive cells in spleens of nu/nu mice after the age of 2 months when the spleen cells were cultured in a medium containing 5% human plasma or human serum, but not when they were cultured in fetal calf serum. Cytotoxic test using anti-Thy-1.2 antibody plus complement has revealed that these Con A-responsive cells are indeed Thy-1+. The Con A-responsive T cells increase with age. PHA-responsive and alloreactive T cells are also detected in the spleens of 11-month-old nu/nu mice. To probe the origins of the T-lymphocytes of the nu/nu mice we examined extensively and systematically the thymic remnants in the mediastinum and neck region of the nu/nu mice in search for T cell development in thymic rudiments. In these investigations we regularly found small accumulations of cells comprising almost entirely Thy-1+ lymphocytes surrounding accumulations surrounded by epithelial cells. These findings suggest that apparent sites of T cell development in thymic rudiments are indeed present in nude mice and appear to be functioning to induce expression T cell markers on precursor cells in the lymphoid lineage. Together with our prior findings the evidence presented in this report shows that a pathway exists in nude mice. In the converse these findings suggest that the thymus is the sole site for induction of the differentiation of stem cells or precursor T cells into mature T-lymphocytes. The findings suggest that indications of alternative differentiation pathways for T-lymphocytes must seek the location of these pathways within the thymus itself.