Activation of calcium-activated, phospholipid-dependent protein kinase C by phorbol esters such as 12–0–tetradecanoyl-phorbol-13–acetate (TPA) has been shown to mediate release of hormones in many systems. To investigate the role of protein kinase C in the mechanism of pituitarv ACTH release, we studied the effect of the following conditions on TPA mediated ACTH release in primary rat pituitary cultures; corticotropin releasing hormone (CRH), different concentrations of extracellular calcium (Ca+2), nifedipine (a calcium channel blocker), PGE2and cortisol (regulators of ACTH secretions). TPA induced ACTH release in a dose dependent fashion with an ED50of 4.2×10–10M. The maximal amount of ACTH release individually induced by TPA (10–8M) and CRH (10–8) were identical. TPA (10–8) potentiated the amount of ACTH release from cells already maximally stimulated with CRH (4×10–8M). TPA mediated ACTH release was dependent on extracellular calcium and inhibited by nifedipine, to a maximum of 35%. Cortisol decreased the amount of ACTH individually released by TPA and CRH in a similar and dose dependent fashion with maximal inhibition of 47% occurring at 10–7M. PGE2caused a dose dependent reduction of TPA mediated ACTH release. In conclusion, the pathways of ACTH release induced by CRH and TPA are not entirely the same but may share a common regulatory pathway. Extracellular calcium and calcium cell influx may be important for maximal ACTH release induced by TPA. Protein kinase C activation may play an important role in CRH stimulated ACTH release.