Experiments were performed in vitro to examine the possible role of calcium and calmodulin in GRF-induced somatostatin (SRIF) release from the median eminence. Adult male rats were used as tissue donors. The median eminences were first prestimulated in 0.4 ml Krebs Ringer bicarbonate glucose buffer (pH 7.4) containing bacitracin at 37C in an atmosphere of 95% O2, 5% CO2 with constant shaking for 30 min. When calcium was omitted, this medium was used during the prestimulation and stimulation periods. After prestimulation, the medium was discarded and replaced by medium containing the different substances to be tested (GRF, EGTA, calcium channel blockers, and calmodulin inhibitors). The stimulation of SRIF release induced by 10(-10) M GRF was not inhibited by omission of extracellular calcium or when the remaining CA+2 was chelated with 10(-4) M EGTA. The calcium channel blockers, nifendipine and verapamil (10(-6) M), failed to alter the increase of SRIF release induced by rGRF. Three calmodulin inhibitors were employed to examine the possible influence of calmodulin on GRF-induced SRIF release. Trifluoperazine (10(-6) M), triflupromazine (10(-6) M) and penfluridol (10(-7) M) had an inhibitory effect on the stimulation of SRIF release induced by GRF and failed to alter resting release. Thus, GRF can evoke SRIF release independently of extraterminal Ca+2 concentration and Ca+2 influx into the nerve terminals, but the releasing process involves translocation of Ca+2 from intracellular stores. The inhibitory effect of the calmodulin inhibitors on GRF-induced SRIF release, suggests that the translocated Ca+2 must bind to calmodulin in order to release SRIF.