20-Hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) is a principal arachidonic acid (AA) metabolite formed via P450-dependent oxidation in hepatic and renal microsomes. Although 20-HETE plays an important role in the regulation of cell and/or organ physiology, the P450 enzyme(s) catalyzing its formation in humans remain undefined. In this study, we have characterized AA omega-hydroxylation to 20-HETE by human hepatic microsomes and identified the underlying P450s. Analysis of microsomal AA omega-hydroxylation revealed biphasic kinetics (KM1 and VMAX1 = 23 microM and 5.5 min-1; KM2 and VMAX2 = 144 microM and 18.8 min-1) consistent with catalysis by at least two enzymes. Of the human P450s examined, CYP4A11 and CYP4F2 were both potent AA omega-hydroxylases, exhibiting rates of 15.6 and 6.8 nmol 20-HETE formed/min/nmol P450, respectively. Kinetic parameters of 20-HETE formation by CYP4F2 (KM = 24 microM; VMAX = 7.4 min-1) and CYP4A11 (KM = 228 microM; VMAX = 49.1 min-1) resembled the low and high KM components, respectively, found in liver microsomes. Antibodies to CYP4F2 markedly inhibited (93.4 +/- 6%; n = 5) formation of 20-HETE by hepatic microsomes, whereas antibodies to CYP4A11 were much less inhibitory (13.0 +/- 9%; n = 5). Moreover, a strong correlation (r = 0.78; P < .02) was found between microsomal CYP4F2 content and AA omega-hydroxylation among nine subjects. The correlation (r = 0.76; P < .02) also noted between CYP4A11 content and 20-HETE formation stemmed from the relationship (r = 0.83; P < . 02) between hepatic CYP4A11 and CYP4F2 levels in the subjects. Finally, immunoblot analysis revealed that in addition to liver, both P450s also were expressed in human kidney. Our results indicate that AA omega-hydroxylation in human liver is catalyzed by two enzymes of the CYP4 gene family, namely CYP4F2 and CYP4A11, and that CYP4F2 underlies most 20-HETE formation occurring at relevant AA concentrations.