The thymic medulla is a complex microenvironment which plays a crucial role in central tolerance induction. Using a quantitative histological analysis of non-obese diabetic (NOD) mice, we show that the medulla undergoes several structural modifications during the course of the disease in NOD mice. Indeed, the majority of 70-day-old NOD mice show a scattering of medullary epithelial cells in the cortex which is associated with a reduction in the size of the medulla in heavily disorganized thymuses. The severity of this phenotype is shown to correlate with the subsequent appearance of diabetes in older female NOD mice. This trait is mainly controlled by non-major histocompatibility complex NOD genes since C57BL/6 H-2g(7) congenic mice have a normal medulla. It persists in conditions where effector lymphocytes that lead to diabetes are inhibited in periphery. These results suggest that primary alterations of the thymic stroma might play a role in the progression towards diabetes in NOD mice.