Islets of Langerhans consist of four major endocrine cell types assembled in a highly organized manner critical for their function. The molecular forces governing islet cellular architecture are not understood. We determined whether adult rat islet cells carry information necessary for orderly assembly. Dispersed cells from adult rat islets were maintained in static suspension culture for 6 days. During this time the cells reassociated to form numerous aggregates. These aggregates were approximately half the size of native islets with a commensurate reduction in DNA and insulin content. However, both cellular composition and organization were remarkably similar to that of adult rat islets, in which the beta-cells form a central core surrounded by a discontinuous mantle of non-beta-cells. Thus, immunoperoxidase staining showed that in the aggregates, just as in intact islets cultured in parallel, 26% of the cells were non-beta-cells and of these, 94% were clearly peripheral. Non-beta-cells were similarly found to be peripheral, with beta-cells located centrally, even when the ratio of non-beta-cells to beta-cells had been altered. This was achieved by sorting the two cell populations by fluorescence-activated flow cytometry, resulting in aggregates with 79% non-beta-cells and 21% beta-cells. Insulin release from the aggregates was stimulated approximately ninefold by raising glucose from 50 to 300 mg/dl, which was comparable to that found for intact islets. The spontaneous formation of isletlike aggregates displaying appropriate cellular architecture indicates that the signals (molecules) needed for such organization are intrinsic to islet cells and are still expressed by them in adult life.