A prospective study of a normal childhood population identified 44 islet cell antibody positive individuals. These subjects were typed for HLA DR and DQ alleles and investigated for the presence of antibodies to the Mr 64,000 (64K) islet cell antigen, complement-fixing islet cell antibodies and radiobinding insulin autoantibodies to determine their potency in detecting subjects with impaired Beta-cell function. At initial testing 64K antibodies were found in six of 44 islet cell antibody positive subjects (13.6%). The same sera were also positive for complement-fixing islet cell antibodies and five of them had insulin autoantibodies. During the follow-up at 18 months, islet cell antibodies remained detectable in 50% of the subjects studied. In all six cases who were originally positive, 64K antibodies were persistently detectable, whereas complement-fixing islet cell antibodies became negative in two of six and insulin autoantibodies in one of five individuals. HLA DR4 (p < 0.005) and absence of asparic acid (Asp) at position 57 of the HLA DQ chain (p < 0.05) were significantly increased in subjects with 64K antibodies compared with control subjects. Of 40 individuals tested in the intravenous glucose tolerance test, three had a first phase insulin response below the first percentile of normal control subjects. Two children developed Type 1 (insulin-dependent) diabetes mellitus after 18 and 26 months, respectively. Each of these subjects was non-Asp homozygous and had persistent islet cell and 64K antibodies. We conclude that 64K antibodies, complement-fixing islet cell antibodies and insulin autoantibodies represent sensitive serological markers in assessing high risk for a progression to Type 1 diabetes in islet cell antibody positive non-diabetic individuals.