Cytochalasin B (10 mug/ml) facilitated glucose-, glyceraldehyde-, and leucine-induced insulin release. It inhibited glucose uptake, utilization, and oxidation, as well as lactate output in islets exposed to glucose (16.7 mM). However, it failed to affect lactate output in the presence of glyceraldehyde, and leucine oxidation. Cytochalasin B also caused a partial inhibition of 45calcium uptake and proinsulin synthesis evoked by glucose in low concentration (5.6 mM), these findings being compatible with a modest impairment of the process of glucose recognition by the beta-cell. At a higher glucose level (16.7 mM), cytochalasin B failed to affect proinsulin synthesis, the immediate inhibitory effect of glucose upon 45calcium efflux, and the subsequent accumulation of 45calcium in the islets. In the presence of cytochalasin B, mannoheptulose further reduced glucose utilization and lactate production and suppressed glucose-induced insulin release. These data suggest that, although insulin release in the presence of cytochalasin B apparently remains dependent on a sufficient glycolytic flux, the facilitating effect of the drug upon insulin secretion cannot be ascribed to any favorable influence on glucose handling by islet tissue. It is suggested that cytochalasin B, possibly through its effect on the microfilamentous web which is part of the cell boundary, may both facilitate insulin release and inhibit glucose transport across the cell membrane, although no direct cause and effect relationship would exist between the two phenomena.