Pancreatic islets of neonatal were dissociated by collagenase and cultured for 3 days in the presence of Cytodex beads to allow attachment of the cells to these microcarriers. The bead-attached cells were packed in columns and superfused with a low bicarbonate medium using the same method that we had originally developed for dissociated anterior pituitary cells of the rat. The secretion of insulin, somatostatin, and glucagon by the cells was monitored by radioimmunoassays. The cells in the superfusion system responded as expected from experiments with islet and monolayer cultures of rat pancreas in vitro and in vivo. Increasing glucose concentrations in the superfusion medium increased the release of insulin and somatostatin (SS), whereas glucagon secretory rates remained constant or decreased. A dose-response curve was established between insulin release and D-glucose in which the ED50 of D-glucose for insulin was found between 1.5 and 2 mg/ml. The phosphodiesterase inhibitor, 3-isobutyl-methylxanthine (IBMX), significantly potentiated the insulin response to glucose. Various secretagogues such as IBMX, 8-bromo cyclic AMP, and L-arginine increased insulin, somatostatin, and glucagon secretory rates in an expected manner. The superfusion method offers the possibility to investigate the interactions of dissociated A-, B-, and D-cells and the dynamics of hormone release in short- and long-term in vitro experiments. The method is simple and avoids the problems of monolayer procedures, such as clustering of the cells and poor adherence of dissociated pancreatic islet cells to dishes.