The effect of cytochalasin B on the biphasic pattern of insulin secretion was studied. Islets were isolated from the normal rat pancreas by the collagenase technique and maintained in vitro by perifusion. Cytochalasin B produced a significant increase in the rate of insulin secretion in the first and second phase of glucose-induced insulin release and enhanced significantly the monophasic pattern of release induced by tolbutamide. The drug had no effect on insulin release in the presence of non—stimulatory levels of glucose. The enhancing effect on glucose—induced insulin secretion required the continued presence of cytochalasin B. Removal of the drug during the second phase of glucose—induced insulin secretion resulted in a rapid decline in the rate of insulin release. Islets were perifused with glucose, glucagon and theophylline in order to obtain maximal stimulation of insulin release. Under these conditions of marked stimulation, cytochalasin B did not produce a further significant enhancement of insulin release. The integrity of the microtubular system was apparently required for the enhancing effect of cytochalasin B since perifusion with vinblastine inhibited the stimulatory effect of glucose and cytochalasin B on insulin secretion. (Endocrinology92: 1458, 1973)