A series of studies was designed to determine the effects of protein kinase C activators on TSH, LH, and GH release from anterior pituitary cells. A 15-min incubation of cultured pituitary cells with synthetic diacylglycerol or phorbol myristate acetate, stimulators of protein kinase C, increased GH, LH, and TSH release. Similarly phospholipase C, which liberates endogenous diacylglycerol, stimulated GH, LH, and TSH secretion. The potentiation of the effects of protein kinase C activators is achieved by calcium mobilization in various cell types. The results of the present studies show that calcium ionophore A23187 or calcium channel activator maitotoxin potentiate diacylglycerol-, phorbol ester-, or phospholipase C-induced GH, LH, or TSH release. These findings suggest that activation of protein kinase C by diacylglycerol and mobilization of calcium may be synergistically involved in the regulation of GH, LH, and TSH release.