Human liver microsomal fractions metabolized arachidonic acid in the presence of NADPH yielding epoxyeicosatrienoic acids and their hydration products, dihydroxyeicosatrienoic acids, as the principal reaction products. Inhibition studies using polyclonal antibodies prepared against recombinant CYP2C8, an abundant human liver cytochrome P450 epoxygenase, demonstrated 85-90% inhibition of arachidonic acid epoxide formation. Both epoxyeicosatrienoic acids and dihydroxyeicosatrienoic acids were detected in large amounts in human liver using gas chromatography/mass spectrometry. Chiral analysis of the endogenous liver epoxides demonstrated a preference for the 14(R),15(S)-, 11(R),12(S)-, and 8(S),9(R)-epoxyeicosatrienoic acid enantiomers. Importantly, the chirality of liver epoxyeicosatrienoic acids matched that previously reported for recombinant CYP2C8 (Zeldin et al. (1995) Arch. Biochem. Biophys. 322, 76-86). Incubations of both human liver cytosolic and microsomal fractions with synthetic epoxyeicosatrienoic acids revealed a 10-fold higher rate of dihydroxyeicosatrienoic acid formation with cytosolic relative to microsomal fractions. Recombinant human liver cytosolic epoxide hydrolase rapidly and regioselectively hydrated epoxyeicosatrienoic acids. We conclude, based on these data, that CYP2C8 is one of the primary, constitutive hepatic arachidonic acid epoxygenases responsible for formation of epoxyeicosatrienoic acids and that cytosolic epoxide hydrolase is the principal liver enzyme which forms the dihydroxyeicosatrienoic acids. We speculate that these biologically active eicosanoids may be important in maintaining homeostasis in the liver.