The nonobese diabetic (NOD) mouse spontaneously develops insulin dependent diabetes mellitus. The disease is associated with a leucocytic infiltration of the pancreatic islets of Langerhans and it is believed that during the development of autoimmune diabetes, the insulin-secreting islet P-cells are destroyed by autoreactive T lymphocytes. We investigated the alteration of lymphocyte subsets in central and peripheral lymphoid organs of NOD female mice with increasing age beginning before the onset of insulitis and ending well after the onset of diabetes. The spleen, inguinal and pancreatic lymph nodes all increased in cell number, especially after the onset of insulitis (8 weeks), and all decreased after the onset of diabetes. Flow cytometric studies showed a widening of the visible side scatter profile of female NOD lymph node cells which coincided with the initiation of insulitis. Anti-CD4 and anti-CD8 double staining of thymocytes revealed a large increase in the double negative population and a corresponding decrease in the double positive population, but this occurred long after the onset of diabetes. Generally, there was an increase in the CD4 : CD8 ratio in the peripheral lymphoid organs during the onset of insulitis which was largely due to an increase in the CD4 T cell population while the ratio decreased after the onset of diabetes. In the spleen this was mostly due to an increase in CD8 T cells. The pancreatic lymph nodes, which theoretically might reflect what is happening in the pancreas, showed an unexpected decrease in overall cell number and a decrease in T-cells (especially CD4 T cells), while B cells were increased. Overall, it would appear that no single immunological parameter could be used to predict a prediabetic state.