Nitric oxide (NO) regulates renal O₂ consumption, but the source of NO mediating this effect is unclear. We explored the effects of renal NO production on O₂ consumption using renal cortex from mice deficient (−/−) in endothelial (e) nitric oxide synthase (NOS). O₂consumption was determined polarographically in slices of cortex from control and eNOS(−/−) mice. NO production was stimulated by bradykinin (BK) or ramiprilat (Ram) in the presence or absence of an NOS inhibitor. Basal O₂ consumption was higher in eNOS(−/−) mice than in heterozygous controls (919 ± 46 vs. 1,211 ± 133 nmol O₂ · min⁻¹ · g⁻¹;P < 0.05). BK and Ram decreased O₂consumption significantly less in eNOS(−/−) mice [eNOS(−/−): BK −19.0 ± 2.8%, Ram −20.5 ± 3.3% at 10⁻⁴ M; control: BK −29.5 ± 2.5%, Ram −34 ± 1.6% at 10⁻⁴ M]. The NO synthesis inhibitor nitro-l-arginine methyl ester (l-NAME) attenuated this decrease in control but not eNOS(−/−) mice. An NO donor inhibited O₂ consumption similarly in both groups independent of the presence of l-NAME. These results demonstrate that NO production by eNOS is responsible for regulation of renal O₂ consumption in mouse kidney.