We tested the possibility that the activation of protein kinase-C by the tumor-promoter phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) or diacylglycerol stimulates the production of prostaglandin E2 (PGE2) by the amnion. Confluent primary cultures of human amnion epithelial cells were adapted to serum-free medium and treated with the agonists for up to 8 h. Cumulative PGE2 output in the medium was measured by RIA. TPA, a potent activator of protein kinase-C, stimulated basal PGE2 output from less than 50 pg/well.5 h to 3 ng/well.5 h (P less than 0.01) in a time- and dose-dependent manner. 4-Methoxy-TPA, a weak tumor promoter derivative of TPA, was ineffective when tested in the same concentration range as TPA (1 nmol/L to 1 mumol/L). Neither calcium ionophore A23187 (20 nmol/L) nor arachidonate (1 mumol/L) stimulated PGE2 output alone, but each agonist potentiated the effect of TPA as much as 5-fold (P less than 0.01). 1,2-Dioctanoyl-sn-glycerol stimulated PGE2 output 4- to 7-fold (P less than 0.05), and this effect was potentiated by Ca ionophore and arachidonate. Studies involving actinomycin-D and cycloheximide indicated that the stimulatory effect of TPA was dependent on RNA synthesis during the first 60 min and on protein synthesis during the entire length of the phorbol ester treatment period (300 min). TPA was also able to stimulate PGE2 production after irreversible inactivation of PG endoperoxide synthase activity with acetylsalicylic acid. These results suggest that activation of protein kinase-C in amnion cells increases the de novo synthesis of the PG endoperoxide synthase enzyme in a RNA synthesis-dependent manner. Elevated intracellular calcium levels contribute to the stimulation apparently by increasing the availability of endogenous arachidonate for subsequent conversion to PGE2.