The aims of our study were to correlate the specificities of islet cell cytoplasmic autoantibodies (ICA) observed in 14 first-degree relatives of diabetic patients with the development of clinical diabetes. To detect whether ICAs recognized in an homogeneous fashion the two major islet cell components, namely beta and alpha cells, the number of islet cells simultaneously recognized by ICAs and anti-glucagon antibodies was determined using a four layer indirect immunofluorescence technique and confocal laser microscopy. The different types of pattern, either whole islet cell staining (type I) or beta cell predominant staining (type II), was then correlated to the presence of autoantibodies to glutamate decarboxylase (GAD), as well as to metabolic outcome. Eight relatives (57.2%) had the type I pattern and 6 (42.8%) the type II pattern. Type 1 diabetes occurred in all ICA positive relatives with the type I pattern and in only 2 out of the 6 relatives with the type II pattern. Interestingly, this later pattern was significantly associated with the presence of autoantibodies to GAD although 2 relatives with antibodies with the type I pattern were also positive. Although more effective for 5 sera with the type II pattern, preincubation with rat brain homogenate significantly decreased the titres of 3/6 sera with the type I pattern. Altogether, our results suggest that autoimmunity to beta cells may be associated with antibodies directed to separate autoantigens with different cellular distributions. From these observations, additional autoantigens distinct from glutamate decarboxylase are also certainly involved.