To investigate whether L-arginine/nitric oxide (NO) pathway activated after treatment with lipopolysaccharide (LPS) could relax the gastric fundus smooth muscle, we made functional examinations and measured NO synthase activity by the conversion of radiolabelled L-arginine to L-citrulline in rat gastric fundus strips treated with LPS in vitro. L-arginine caused a relaxation of the mucosa-free gastric fundus strips which had been treated with LPS for 6 h in vitro and then contracted by PGF2alpha beforehand. This relaxation was partially reversed by N(G)-nitro-L-arginine (a nitric oxide synthase inhibitor) or methylene blue (a soluble guanylate cyclase inhibitor). Ca(2+)-independent NO synthase activity was induced after LPS-treatment. Co-incubation with LPS and cycloheximide for 6 h inhibited the relaxation to L-arginine and the induction of NO synthase. On the other hand, Ca(2+)-dependent NO synthase activity was decreased after LPS-treatment. These results strongly suggest that Ca(2+)-independent NO synthase is induced by endotoxin in the gastric fundus muscle, resulting in inhibition of the contractile response.