Markers of humoral islet cell autoimmunity, such as autoantibodies (AAs) against the 65-kDa isoform of GAD (GAD65), serve as determinants of risk for autoimmune diabetes. Despite the high prevalence of diabetes in U.S. racial and ethnic minority adult populations, little is known concerning the prevalence of GAD65 AA in these groups. We estimated the prevalence of GAD65 AA in 1,064 diabetic and 1,036 nondiabetic participants who were 40-90 years of age from the Third National Health and Nutrition Examination Survey (NHANES III), which provides a representative ethnic sample of the U.S. diabetic population. The prevalence of GAD65 AA was higher in diabetic participants compared with nondiabetic participants in non-Hispanic whites (n = 920; 6.3% vs. 2.0%; P = 0.001) and non-Hispanic blacks (n = 534; 3.7% vs. 1.3%; P = 0.08) but not in Mexican Americans (n = 646; 1.2% vs. 2.6%; P = 0.18). Among diabetic non-Hispanic whites and non-Hispanic blacks, being GAD65 AA positive was associated with lower BMI and C-peptide (P < 0.05). These results may reflect the outcome of an autoimmune process leading to beta-cell destruction/dysfunction in non-Hispanic white and non-Hispanic black adult diabetic patients as it occurs in a similar manner in type 1 diabetes. Among diabetic Mexican Americans, the lower prevalence of GAD65 AA suggests a lower frequency of autoimmune-related diabetes.