In vertebrates, nitric oxide (NO) is synthesized from L-arginine by NO synthase (NOS) and regulates relaxation of smooth muscle by activating the cyclic-GMP (cGMP) generating enzyme soluble guanylyl cyclase (SGC). Here we show that the NO-cGMP pathway mediates relaxation of the cardiac stomach in the starfish Asterias rubens. The NO-donors hydroxylamine, S-nitrosoglutathione (SNOG) and S-nitroso-N-acetylpenicillamine (SNAP) and the NOS substrate L-arginine cause relaxation of the cardiac stomach. The relaxing effect of SNAP is blocked by the SGC inhibitor 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), and the relaxing effect of L-arginine is inhibited by ODQ and the NOS inhibitor Nw-monomethyl-L-arginine (L-NMMA). ODQ and methylene blue also cause contraction, which may be due to inhibition of the relaxing action of NO produced by cells in the cardiac stomach. These results suggest that NO is synthesized in the cardiac stomach and regulates relaxation by activating SGC. NO-cGMP-mediated relaxation of the cardiac stomach may be important during feeding in starfish where the relaxed stomach is everted through an oral opening and over the digestible parts of prey. The discovery of NO-cGMP-mediated relaxation in an echinoderm demonstrates that regulation of smooth muscle tone by this signaling pathway also occurs in animals other than vertebrates.