Co-oxidation of phenacetin, acetaminophen (APAP) and p-aminophenol (p-AP) by prostaglandin H synthase (PHS) was investigated in human and rat renal microsomes. The formation of prostaglandin E2 (PGE2) was assessed in cortex, outer and inner medulla following phenacetin, APAP and p-AP (0-5 mM) incubations. For all compounds and concentrations tested, a significantly higher PGE2 production was observed in inner medulla compared to cortex. Rat inner medulla incubated with phenacetin resulted in an increased formation of PGE2 at all concentrations compared to control (1 mM phenacetin increased production by 243%). Human inner medulla demonstrated an increased PGE2 production at 1, 3 and 5 mM phenacetin versus control (136% increase at 1 mM). An increase in PGE2 formation in rat and human inner medulla was observed at low APAP concentrations (0.1, 0.3, 0.5 and 1 mM) compared to control (216% and 396% in human and rat respectively following 1 mM APAP). 5 mM APAP inhibited PGE2 formation in the rat inner medulla but not in human inner medulla. An inhibition of PGE2 production by 5 mM p-AP was observed in both the rat and human inner medulla. In the rat PGE2 production was inhibited 69% by 5 mM, whereas in the human the inhibition was 76% at 5 mM. These studies demonstrate a species-specific PHS-mediated renal metabolism of APAP, with the human kidney demonstrating a continous formation of reactive metabolites at high concentrations of APAP. However, phenacetin and p-AP are metabolized in a similar manner in these 2 species.