Islet cells of adult rat pancreas, dissociated with EDTA-Dispase, were cultivated in Microtest wells for over 2 months. In our cultures, islet cells were free-floating and cohered with each other to reorganize histotypic aggregates resembling the nondissociated islets. Morphologically, excellent preservation of islet cells in the aggregates was confirmed during the culture using both light and electron microscopy. The function of islet beta-cells, as demonstrated by the synthesis and release of insulin, also was retained throughout the culture period. Islet beta-cells cultured for long periods exhibited better response to the short-term stimulation of theophylline than to a high concentration of glucose, as observed in the islets of fetal or newborn rat pancreas.