The tumor promoters, 12-O-tetradecanoylphorbol-13-acetate (TPA), phorbol-12,13-didecanoate, teleocidin, and dihydroteleocidin, at nM levels, but not the non-tumor-promoting 4 alpha-phorbol-12,13-didecanoate even at microM concentrations, stimulated arachidonic acid metabolism in cultured rat liver cells. These liver cells synthesize primarily prostaglandin I2 [measured as its nonenzymatic hydrolytic product, 6-keto-prostaglandin F1 alpha (PGF1 alpha)]. The production of 6-keto-PGF1 alpha increased with time of incubation with TPA and was essentially complete in 4 hr. Cycloheximide, at nM levels, blocked the TPA-stimulated 6-keto-PGF1 alpha production in a dose-dependent manner; this inhibition was related to inhibition of protein synthesis. Chelation of Ca2+ by ethyleneglycol-bis(beta-aminoethyl ether)-N,N'-tetraacetic acid, treatment of the cells with the Ca2+ channel blocker, nifedipine, or inhibition of intracellular Ca2+ mobilization by 8-(diethylamine)octyl-3,4, 5-trimethoxybenzoate hydrochloride also inhibited TPA-stimulated 6-keto-PGF1 alpha production. The steroidal antiinflammatory drug, dexamethasone, a potent in vivo inhibitor of tumor promotion, was an inhibitor of 6-keto-PGF1 alpha stimulation by TPA.