The effect of NG-nitro-L-arginine (L-NNA) on regional cerebral blood flow (rCBF) response to hypercapnia (5% CO2 inhalation) was studied in urethan-anesthetized wild-type (SV-129) and type III nitric oxide (NO) synthase (NOS)-deficient mice, using laser-Doppler flowmetry and the closed cranial window technique. Resting rCBF during normocapnia decreased by approximately 25% after L-NNA superfusion in wild-type mice only (n = 18), suggesting a role for type III NOS in baseline blood flow. Hypercapnia augmented rCBF approximately 50% in both wild-type and type III NOS mutant mice. L-NNA superfusion (1 mM) inhibited this increase by approximately 60% in both strains. Hence, synthesis of NO by the constitutively expressed type I NOS contributes to blood flow augmentation during hypercapnia.