To answer the question whether insulin or proinsulin would be the true antigen for both insulin and proinsulin autoantibodies, displacement experiments of 125I-insulin and -proinsulin binding with both unlabeled antigens were performed in sera of four groups of antibody-positive probands: first-degree relatives of Type 1 diabetic patients, pre-Type 1 diabetic persons, recent-onset Type 1 diabetic patients, insulin-treated Type 1 diabetic patients. In subjects who were primarily screened to constitute these groups, prevalences of insulin and proinsulin autoantibodies were nearly identical. In antibody-positive sera, 125I-insulin and -proinsulin binding values in general were closely correlated to each other with regression coefficients near 1.0. In all groups of probands, mean values of 125I-insulin and -proinsulin binding did not significantly differ. With the exception of a few sera, insulin and proinsulin antibodies differentiated only little between both antigens. Epitopes of the insulin molecule are therefore preferred. Nevertheless, insulin and proinsulin autoantibodies are not completely identical nor are insulin autoantibodies merely a subgroup of proinsulin autoantibodies: In each group, in the mean, insulin antibodies as well as proinsulin antibodies reacted somewhat (but significantly) stronger with their respective antigen. In some cases a distinct (relative) specificity for either antigen of insulin and proinsulin autoantibodies were observed, the latter being still present after some months of insulin treatment. In conclusion, despite detectable differences in antigen specificity, insulin and proinsulin autoantibodies seem to be equally potent markers of Type 1 diabetes mellitus.