In nonobese diabetic (NOD) mice, type I diabetes can be prevented without generalized immunosuppression by nonhypercalcemic analogs of vitamin D3 when treatment is started early, i.e. before the autoimmune attack, reflected by insulitis, occurs. The aim of this study was to investigate whether these substances can arrest progression to clinically overt diabetes when administered in a more advanced disease stage, namely when the autoimmune attack is ongoing, reflecting the situation in prediabetic subjects in whom immune intervention is being considered. We, therefore, evaluated the protective potential of MC1288 (20-epi-1,25-dihydroxyvitamin D3) a nonhypercalcemic analog of 1,25-dihydroxyvitamin D3, both alone and in combination with a short induction course of cyclosporin A, in NOD mice that already have insulitis, as demonstrated in pancreatic biopsies performed 15 days before the start oftherapy. Subsequently, mice were randomized into a control group, receiving the treatment vehicle (n = 26), and three treatment groups, receiving, respectively, 7.5 mg/kg x day cyclosporin A (CyA) from days 85-105 (n = 19), 0.1 microg/kg x 2 days MC1288 from days 85-200 (n = 20), or the combination of these two regimens (n = 20). At the time of the pancreatic biopsy (day 70), insulitis was evenly distributed in all groups, and 27.7% of the islets scored showed signs of destructive insulitis. Diabetes outcome by 200 days was 74% (14 of 19) in the CyA-treated group, comparable to the diabetes incidence in control mice (65%; 17 of 26; P = NS). Treatment with MC1288 alone could not reduce disease incidence (70%; 14 of 20), but the combination therapy reduced diabetes incidence to 35% (7 of 20; P < 0.05 vs. untreated; P < 0.01 vs. CyA group; P < 0.025 vs. MC1288). All treatments were well tolerated, without major side-effects on calcium or bone metabolism and without signs of generalized immunosuppression. Cotransfer experiments could not reveal the induction of suppressor cells. Reverse transcription-PCR on pancreatic tissue revealed significantly lower levels of interferon-gamma and higher levels of interleukin-4 in the combination group. In conclusion, nonhypercalcemic analogs of 1,25-dihydroxyvitamin D3 administered to NOD mice when the autoimmune disease is already active can prevent clinical diabetes when this therapy is combined with a short induction course of an immunosuppressant such as CyA.