These studies were designed to examine the effects of extracellular calcium ion (Ca++) concentration upon basal and dibutyryl (db) cAMP or potassium ion (K+)-stimulated release of growth hormone (GH) and to determine whether increased extracellular Ca++ can overcome somatostatin (SRIF)-inhibited release of stored rGH in parallel with its reported effect upon SRIF inhibition of stimulated insulin and glucagon release. Experiments were performed in vitro using prelabeled rat pituitary fragments in a perifusion, specific immunoprecipitation system designed to limit observations to release of stored hormone from viable cells. Increased (up to 5.4 mM) extracellular Ca++ inhibits basal and dbcAMP-stimulated release of stored, prelabeled [3H]rGH in parallel with the effects of SRIF: post-inhibition rebound, dose responsivity, and differential effect upon early and late dbcAMP-stimulated release of stored [3H]rGH. Increased (21 mM) extracellular K+ interferes with both Ca++- and SRIF-inhibited early dbcAMP-stimulated release of stored [3H]rGH. The combination of increased extracellular Ca++ and SRIF inhibits basal release of stored [3H]rGH more than either agent alone and during dbcAMP stimulation, rebound release of stored [3H]rGH follows withdrawal of either inhibitor despite continuation of the other. This rebound release is enhanced when both inhibitors are withdrawn simultaneously. CONCLUSIONS (a) the inhibition of stored rGH release induced by increased extracellular Ca++ and SRIF occurs through at least partially independent mechanisms, and (b) increased extracellular Ca++ does not reverse SRIF inhibition of stimulated rGH release from prelabeled intracellular storage, in contrast with observations in the pancreatic islet.