Nitric oxide (NO) is a potent vasodilator in terrestrial vertebrates, but whether vascular endothelial‐derived NO plays a role in vascular regulation in fish remains controversial. To explore this issue, a study was made of spiny dogfish sharks (Squalus acanthias) in normoxia and acute hypoxia (60 min exposure to seawater equilibrated with 3% oxygen) with various agents known to alter NO metabolism or availability. In normoxia, nitroprusside (a NO donor) reduced blood pressure by 20%, establishing that vascular smooth muscle responds to NO. L‐arginine, the substrate for NO synthase, had no hemodynamic effect. Acetylcholine, which stimulates endothelial NO and prostaglandin production in mammals, reduced blood pressure, but also caused marked bradycardia. L‐NAME, an inhibitor of all NO synthases, caused a small 10% rise in blood pressure, but cell‐free hemoglobin (a potent NO scavenger and hypertensive agent in mammals) had no effect. Acute hypoxia caused a 15% fall in blood pressure, which was blocked by L‐NAME and cell‐free hemoglobin. Serum nitrite, a marker of NO production, rose with hypoxia, but not with L‐NAME. Results suggest that NO is not an endothelial‐derived vasodilator in the normoxic elasmobranch. The hypertensive effect of L‐NAME may represent inhibition of NO production in the CNS and nerves regulating blood pressure. In acute hypoxia, there is a rapid up‐regulation of vascular NO production that appears to be responsible for hypoxic vasodilation. J. Exp. Zool. 303A:154–160, 2005. © 2005 Wiley‐Liss, Inc.