A quantitative fluid-phase radioassay for autoantibodies reacting with insulin (competitive insulin autoantibody assay, CIAA) was developed. The assay's features include 1) use of a physiologic amount of ¹²⁵I-labeled insulin, 2) parallel incubations with supraphysiologic cold insulin (competitive), and 3) an incubation time of 7 days and a single-step multiple-wash polyethylene glycol separation. Mean ± SE CIAA levels in 50 controls were 8 ± 1.4 nU/ml (range −16–33.3). In 36 cytoplasmic islet cell antibody (ICA)-positive nondiabetic first-degree relatives of type I (insulin-dependent) patients <30 yr of age, CIAA levels exceeded the normal range in 20 (55.6%) of 36 (mean 86.8 ± 17.1 nU/ml). In 26 ICA-positive relatives >30 yr of age, only 5 (19.2%) of 26 exceeded the normal range (mean 26.1 ± 9.4 nU/ml); P < .001 compared with younger ICA-positive relatives). Six ICA-negative HLA-identical siblings of type I diabetic patients had normal CIAA levels (mean 3.6 ± 5.8 nU/ml), and only 2 of 13 ICA-negative identical twins discordant for diabetes (mean 15.4 ± 6.6 nU/ml) exceeded the normal range. Nine (50%) of 18 ICApositive schoolchildren exceeded the normal range (mean 105.3 ± 36.7 nU/ml). Genetically susceptible subjects negative for CIAA (with only 3 exceptions) remained negative for CIAA on multiple determinations (3 conversions observed), and CIAA levels of positive subjects were relatively stable. Linear regression of the first CIAA level versus last (interval between sampling 1 mo to 10 yr) in genetically susceptible individuals showed a highly significant correlation (r = .95, P < .001). This simple radioimmunoassay appears to have requisite specificity and sensitivity to allow screening populations at risk to develop type I diabetes.