The objective of this study was to determine the extent to which different screening strategies could identify a population of nondiabetic relatives of a proband with type 1 diabetes who had two or more immunologic markers from the group consisting of islet cell antibodies (ICA), micro insulin autoantibodies (MIAA), GAD65 autoantibodies (GAA), and ICA512 autoantibodies (ICA512AA). Relatives of subjects with type 1 diabetes were screened for ICA as part of the Diabetes Prevention Trial-Type 1. A total of 71,148 samples were also tested for GAA and ICA512AA. IAA results were available on 17,207 of these samples using a protein A/protein G MIAA assay as well. The study population was defined to be those in which all four antibodies were tested. There were 1010 (5.9%) relatives with a single autoantibody on initial screening and 394 (2.3%) with two or more autoantibodies. GAA was more sensitive than ICA [GAA, 91% (357 of 394); ICA, 82% (324 of 394)] in the detection of multiple antibody-positive individuals. The addition of ICA512AA to GAA as a screening test increased sensitivity to 97% (381 of 394), whereas adding ICA512AA to ICA as a screening test increased sensitivity to 93% (367 of 394). GAA and ICA identified somewhat nonoverlapping subgroups of multiple antibody-positive subjects. Thus, the substitution of GAA or ICA for the other failed to detect 8-17% of multiple antibody subjects. Higher ICA titers were associated with increased percentages of multiple antibody-positive subjects; 86% of subjects having Juvenile Diabetes Foundation titers of at least 160 were positive for two or more antibodies. A screening strategy combining GAA and ICA512AA resulted in a higher sensitivity than using any marker individually, although statistically it was not significantly higher than using GAA alone. Screening for any three antibodies guaranteed that all multiple antibody-positive subjects were detected. Screening for two antibodies at one time and testing for the remaining antibodies among those who are positive for one resulted in a sensitivity of 99% for GAA and ICA, 97% for GAA and MIAA or GAA and ICA512AA, 93% for ICA512AA and ICA, 92% for MIAA and ICA, and 73% for ICA512AA and MIAA. From a laboratory perspective, screenings for GAA, ICA512AA, and MIAA are semiautomated tests with high throughput that, if used as initial screen, would identify at first testing 67% of the 2.3% of multiple antibody-positive relatives (100% if antibody-positive subjects are subsequently tested for ICA) as well as 4.7% of relatives with a single biochemical autoantibody, some of whom may convert to multiple autoantibody positivity on follow-up. Testing for ICA among relatives with one biochemical antibody would identify the remaining 33% of multiple antibody-positive relatives. Further follow-up and analysis of actual progression to diabetes will be essential to define actual diabetes risk in this large cohort.