Nonobese dibetic (NOD) mice aged 10 weeks were intravenously treated with choleratoxin at a dose of 1.0 μg every two weeks until 24 weeks of age. At the age of 25 weeks, diabetes developed in 13 of 18 saline-treated control NOD mice. These mice showed partial or complete destruction of islets of Langerhans associated with marked lymphocytic infiltration. In contrast, diabetes was observed in 4 of 16 choleratoxin-treated NOD mice and the pancreatic islets showed only mild insulitis. Moreover, two-color flow cytometry analysis of T-cell subsets in peripheral blood revealed significant decreases in the percent of L3T4+ cells and Lyt 2+ cells in choleratoxin-treated NOD mice compared with saline-treated control NOD mice. These results clearly indicate that choleratoxin prevents diabetes mellitus in NOD mice by inhibiting the development of insulitis by interfering with T-cell proliferation.