Electrophysiological studies of rat islet cells in monolayer culture were undertaken to determine the role of transmembranous ionic fluxes in the inhibitory action of somatostatin on insulin release. In the presence of somatotropin release inhibiting factor (SRIF) (2.5 nM), hyperpolarization occured with or without glucose (16.6 mM) in the medium. SRIF also inhibited the incidence of glucose-induced spike activity. The inhibitory action of SRIF occurred within 5 min and was readily reversible. An increase in extracellular K+ (5-13 mM) or Ca2+ (2.3-4.6 mM) prevented SRIF inhibition of glucose-induced electrical activity. The secretory response of cultured islets to glucose (16.6 mM) was completely inhibited by SRIF (2.5 nM). The presence of high [Ca2+]o or [k+]o enhanced insulin release in the presence of SRIF and glucose. Although phentolamine (5.0 microgram/ml) did not block the inhibition of glucose-induced electrical responses by SRIF, it prevented the inhibitory action of epinephrine (0.2 microgram/ml). It is concluded that the primary action of SRIF is to alter transmembranous cationic fluxes, as manifested by hyperpolarization and a decrease in the incidence of spike activity, which may prevent glucose from eliciting a normal secretory response.