The nonobese diabetic (NOD) mouse develops an autoimmune type I diabetes, which is predominantly seen in females, is triggered by T cells, and whose frequency is enhanced following thymectomy at weaning. Attempting to characterize a thymic pathology in these animals, we analyzed the microenvironmental compartment of the organ with respect to structural and functional molecules expressed by thymic epithelial cells (TEC), as well as extracellular matrix components. We observed, in both males and females, a precocious decrease in the cell numbers of discrete medullary TEC subsets, namely, those respectively defined by the expression of cytokeratins 3/10 and cytokeratin 19. In addition, some cells bearing the TR.5 phenotype (normally restricted to the medulla) could be detected in the NOD mouse thymic cortex. There was also a significant early decrease in thymulin production in females, as compared to males. As regards the extracellular matrix compartment, the most striking alteration was the presence of abnormally enlarged perivascular spaces, increasing in size with age. In these structures large amounts of T cells and, to a lesser extent, B cells were consistently encountered. In addition to B cells, the NOD mouse thymus showed on both TEC and extracellular matrix the presence of deposits of immunoglobulins, revealed with fluorescence-labeled goat anti-mouse Ig sera. Finally, the NOD mouse sera labeled both TEC and extracellular matrix proteins on normal mouse thymus frozen sections. Together, these data clearly demonstrate that the NOD mouse thymus undergoes a variety of microenvironmental changes, whose particular role in the pathophysiology of the disease is yet to be demonstrated.