To determine the best autoantibody-based testing strategy for recruiting relatives for future intervention trials and to establish the role of islet cell antibodies (ICAs) within this strategy. ICAs, insulin autoantibodies (IAAs), GAD antibodies, and IA-2 antibodies were determined in serum samples at study entry of 3,655 non-diabetic first-degree relatives of patients with type 1 diabetes who were followed for a median of 5.5 years. The cumulative risk of diabetes associated with single and combined antibody marker levels of > or = 97.5th percentile in schoolchildren was calculated by using life-table analysis. Of the 26 relatives who developed insulin-requiring diabetes during follow-up, 16 were aged < 20 years and 7 were aged 20-39 years at study entry. Of the 23 cases aged < 40 years, 83% had IA-2 and/or GAD antibodies, and 87% had IAA and/or GAD antibodies > or = 97.5th percentile compared with 61% who had ICAs of > or = 5 Juvenile Diabetes Foundation units (JDF U). A two-step strategy with parallel testing for IA-2/GAD antibodies followed by IAA testing identified 50% of cases aged < 20 years and was associated with a 71% risk within 10 years. In subjects aged 20-39 years, this strategy conferred a 51% risk, whereas using ICAs as the second test gave 86% sensitivity and a 74% risk. Primary screening for IA-2 and/or GAD antibodies followed by testing for IAA and/or ICA antibodies achieved the highest sensitivity in both age-groups and conferred a 63% risk. In contrast, ICAs of > or = 20 JDF U (the inclusion criteria for the European Nicotinamide Diabetes Intervention Trial) gave 48% sensitivity and 35% risk. ICA testing can be replaced as a primary screening measure by IA-2/GAD or IAA/GAD antibody testing. The sensitivity of ICAs (used alone or in combination with IAAs) gives them a useful role in second-line testing. Combination testing could reduce the size of screening populations needed for recruitment in future intervention trials by approximately 50% compared with testing based on ICAs alone.