The effects of and interactions between the major phenytoin (PHT) metabolite 5-parahydroxyphenyl-5-phenylhydantoin (p-HPPH) and interleukin-1 (IL-1 alpha, IL-1 beta) or tumor necrosis factor alpha (TNF alpha) on prostaglandin biosynthesis in human gingival fibroblasts were studied. IL-1 alpha, IL-1 beta and TNF alpha, dose-dependently, stimulated PGE2 formation in gingival fibroblasts. The metabolite, p-HPPH (1.2-2.4 micrograms/ml), did not induce PGE2 formation itself but potentiated IL-1 alpha and IL1 beta induced PGE2 formation in the gingival fibroblasts in a manner dependent on the concentration of both IL-1 and p-HPPH. The metabolite also stimulated IL-1 induced formation of 6-Keto PGF1 alpha, the stable breakdown product of PGI2, in a dose dependent manner. IL-1 beta induces release of [3H]-arachidonic acid ([3H]-AA) from prelabelled fibroblasts, which was potentiated by p-HPPH (> or = 1.2 micrograms/ml). TNF alpha (> or = 1 ng/ml) significantly stimulated the biosynthesis of PGE2 by a process that was also potentiated by p-HPPH. Addition of exogenous, unlabelled AA (10 microM) caused an increase of PGE2 formation in the fibroblasts that was not potentiated by p-HPPH (1.6 micrograms/ml). The results indicate that treatment with p-HPPH results in upregulation of prostaglandin synthesis in gingival fibroblasts challenged to IL-1 or TNF alpha at the level of phospholipase A2.