Intensive insulin therapy in patients with recently diagnosed insulin-dependent diabetes mellitus (IDDM) has been reported to result in a prolonged increase in endogenous insulin-secreting capacity. Because the clinical onset of IDDM occurs only after most insulinsecreting β-cells have been destroyed, we tested whether prophylactic insulin therapy might prevent IDDM in nonobese diabetic (NOD) mice. One hundred fourteen NOD mice were randomized at weaning into a protamine zinc pork insulin-treated (I) group or a placebo-treated (P) group given insulin diluent. All insulin treatments were adjusted to the maximum tolerable dosages and continued until 180 days of age. The cumulative IDDM frequency within the female I group was significantly less (3 of 34, 8%) than in female P controls (17 of 26, 65%; P < 0.0001). This beneficial effect was limited to females, however, because the frequency of IDDM in male I mice (3 of 32, 9%) was not significantly different from the frequency in male P controls (1 of 22, 5%; P < 0.5). Pancreatic histological examinations of nondiabetic animals revealed that insulin treatment resulted in significant reductions in islet cell inflammation and damage and improvements in insulin content. In summary, NOD mice given insulin therapy from weaning until 180 days of age had significantly lower frequencies of diabetes and pancreatic insulitis than sex-matched control littermates treated with insulin diluent. These results suggest that prophylactic insulin therapy to prevent IDDM in humans should be considered for clinical trials.