We examined whether parenteral regular insulin can prevent diabetes in IA-2 antibody-positive (IA-2A+) relatives of type 1 diabetic patients, using a trial protocol that differed substantially from that of the Diabetes Prevention Trial-1. Twenty-five IA-2A+ relatives received regular human insulin twice a day for 36 months, during which time they were followed (median [interquartile range; IQR]: 47 [19-66] months) for glucose tolerance, HbA(1c) and islet autoantibodies, together with 25 IA-2A+ relatives (observation/control group) who fulfilled the same inclusion criteria, but were observed for 52 [27-67] months (P=0.58). Twelve (48%) insulin-treated relatives and 15 (60%) relatives in the control group developed diabetes. There was no difference in diabetes-free survival between the two groups (P=0.97). Five-year progression (95% confidence interval) was 44% (25-69) in the insulin-treated group and 49% (29-70) in the observation group. At inclusion, progressors tended to have a higher pro-insulin/C-peptide ratio than non-progressors when measured 2 hours after a standardized glucose load (median [IQR]: 2.7% [1.8-4.3] vs. 1.6% [1.1-2.1]; P=0.01). No major hypoglycaemic episodes or significant increases in body mass index or diabetes autoantibodies were observed. Prophylactic injections of regular human insulin were well tolerated, but failed to prevent type 1 diabetes onset in IA-2A+ relatives.