In reptiles, accumulating evidence suggests that nitric oxide (NO) induces a potent relaxation in the systemic vasculature. However, very few studies have examined the source from which NO is derived. Therefore, the present study used both anatomical and physiological approaches to establish whether NO-mediated vasodilation is via an endothelial or neural NO pathway in the large arteries of the estuarine crocodile Crocodylus porosus. Specific endothelial nitric oxide synthase (NOS) staining was observed in aortic endothelial cells following nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and endothelial NOS immunohistochemistry (IHC), suggesting that an endothelial NO pathway is involved in vascular control. This finding was supported by in vitro organ bath physiology, which demonstrated that the relaxation induced by acetylcholine (10(-5) mol l(-1)) was abolished in the presence of the NOS inhibitor, N-omega-nitro-l-arginine (l-NNA; 10(-4) mol l(-1)), the soluble guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10(-5) mol l(-1)), or when the endothelium was removed. Interestingly, evidence for a neural NO pathway was also identified in large arteries of the crocodile. Neural NOS was located in perivascular nerves of the major blood vessels following NADPH-d histochemistry and neural NOS IHC and in isolated aortic rings, l-NNA and ODQ, but not the removal of the endothelium, abolished the relaxation effect of the neural NOS agonist, nicotine (3 x 10(-4) mol l(-1)). Thus, we conclude that the large arteries of C. porosus are potentially regulated by NO-derived from both endothelial and neural NOS.