Nitric oxide (NO) inhibits hemoproteins including cytochrome (CYP)2C, the gene responsible for the production of epoxyeicosatrienoic acids (EETs). EETs and NO are produced in the kidney and both regulate vascular tone and Na⁺ transport. However, the role of EETs in NO-mediated renal function is not known. This study tested the hypothesis that NO tonically regulates the renal production of EETs thereby impacting renal vasomotor tone and electrolyte balance. Lipopolysaccharide (LPS, 10 mg/kg i.v.) inhibited microsomal conversion of ¹⁴C-AA to EETs and reduced mean arterial blood pressure (MABP; = 63±5 mmHg). L-NAME (10 mg/kg), an inhibitor of NO synthase, increased MABP (= 26±6 mmHg) and reduced cortical (CBF; = -0.86±0.15 volts) and medullary blood flow (MBF, = -0.34±0.09 volts), GFR (0.82±0.16 to 0.32±0.10 ml/g kidney/min), and increased Na⁺ excretion (U_NaV, 0.16+0.04 to 0.30+0.06 µmol/g kidney/min). 2-(2-propynyloxy)-benzenehexanoic acid (PPOH), a suicide substrate inhibitor of EET production, did not affect L-NAME-induced increase in MABP but attenuated the effects of L-NAME on CBF (31±7%, p<0.05%), GFR (44±6%; p<0.05), and U_NaV (78±7%; p<0.05). Miconazole (1.3 mg/kg/hr), a heme inhibitor of epoxygenase enzymes, produced similar effects as PPOH. Intrarenal arterial infusion of 5,6-, 8,9-, 11,12-, and 14,15-EET (1-10 ng/min) elicited dose-dependent reductions in CBF and GFR accompanied by regioisomeric changes in MBF, U_NaV and UV. In addition, 11,12-EET dose-dependently restored the PPOH-blunting of L-NAME effects on CBF, MBF, and GFR. We conclude that NO tonically regulates epoxygenase activity and that EETs are renal vaosocontrictors in vivo and contribute at least, in part, to the renal functional response following inhibition of NO production.