The role of different isoforms of nitric oxide synthase (NOS) in the gastric mucosal hyperemia, induced by 155 mM luminal hydrochloric acid (pH ≈ 0.8) without a barrier breaker, was investigated. Rats were anesthetized with Inactin (120 mg/kg ip), and mice were anesthetized with Forene (2.2% in 40% oxygen gas at 150 ml/min); the gastric mucosa was exteriorized. Gastric mucosal blood flow was measured with laser-Doppler flowmetry (LDF) in rats treated with Nω-nitro-l-arginine (l-NNA; unspecific NOS inhibitor), l- N6-(1-iminoethyl)lysine [l-NIL; inducible (i) NOS inhibitor], or S-methyl-l-thiocitrulline [SMTC; neuronal (n) NOS inhibitor], 10 mg/kg, followed by 3 mg · kg–1 · h–1 iv, in iNOS-deficient (–/–) and nNOS(–/–) mice. mRNA was isolated from the gastric mucosa in iNOS(–/–) and wild-type (wt) mice, and real-time RT-PCR was performed. The effect of 155 mM acid on gastric mucosal permeability was determined by measuring the clearance of 51Cr-EDTA from blood to lumen. LDF increased by 48 ± 13% during 155 mM HCl luminally, an increase that was abolished by l-NNA, SMTC, or l-NIL. In iNOS wt mice, LDF increased by 33 ± 8% during luminal acid. The blood flow increase was attenuated substantially in iNOS(–/–) mice. RT-PCR revealed iNOS mRNA expression in the gastric mucosa in the iNOS wt groups. The blood flow increase in response to acid was not abolished in nNOS(–/–) mice (nNOS-sufficient mice, 39 ± 18%; heterozygous mice, 25 ± 19%; –/– mice, 19 ± 7%). Mucosal permeability was transiently increased during 155 mM HCl. The results suggest that iNOS is constitutively expressed in the gastric mucosa and is involved in acid-induced hyperemia, suggesting a novel role for iNOS in gastric mucosal protection.