20-Hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE), the omega-hydroxylation product of arachidonic acid, is the major metabolite produced in the kidney. It has potent biological effects on renal tubular and vascular functions and on the long-term control of arterial pressure. The synthesis of 20-HETE is catalyzed by enzymes of the CYP4A family, among which CYP4A2 is the most abundant isozyme expressed in the kidneys of rats. We have cloned and sequenced the CYP4A2 cDNA from the kidney of Lewis-Wistar rats and directed its expression using baculovirus and Sf9 insect cells. A high level of expression of CYP4A2 was evident by Northern, Western, and spectral analyses revealing a P450 content of 0.3 nmol/mg microsomal protein. To study CYP4A2-catalyzed arachidonic acid omega-hydroxylation, Sf9 cells were coinfected with CYP4A2 and NADPH cytochrome P450 oxidoreductase (OR) recombinant viruses. CYP4A2/OR membranes metabolized lauric acid at a high rate (7 and 5.5 nmol/min/nmol P450 in the presence and absence of b5, respectively). However, arachidonic acid omega-hydroxylase activity was barely detectable. When purified OR was added to the membranes expressing CYP4A2 protein, a concentration-dependent production of 20-HETE was observed. Maximal synthesis of 20-HETE of 0.89 nmol/min/nmol P450 was achieved at OR:CYP4A2 ratio of 14:1. The omega-hydroxylation of arachidonic acid was dependent on the presence of b5. Furthermore, increasing OR concentrations yielded additional arachidonic acid metabolite identified by GC/MS as 11,12-EET. Microsomes prepared from isolated renal microvessels selectively expressed CYP4A2 protein and readily metabolized arachidonic acid to two major metabolites, 20-HETE and 11,12-DHET, the hydrolytic metabolite of 11, 12-EET. It is suggested that CYP4A2 functions as the renal microvessel arachidonate omega-hydroxylase and that it can also catalyze the 11,12-epoxidation of arachidonic acid.