In this study, the role of NO in regulation of the pulmo-cutaneous vasculature of the toad, Bufo marinus, was investigated. In vitro myography demonstrated the presence of a neural NO signalling mechanism in both arteries. Vasodilation induced by nicotine was inhibited by the soluble guanylyl cyclase (GC) inhibitor, 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), and the nitric oxide synthase (NOS) inhibitor, N Nitro L arginine (L-NNA). Removal of the endothelium had no significant effect on the vasodilation. Furthermore, pre-treatment with N⁵-(1-Imino-3-butenyl)-L-ornithine (vinyl-L-NIO), a more specific inhibitor of neural NOS (nNOS), caused a significant decrease in the nicotine-induced dilation. In the pulmonary artery only, a combination of L-NNA and the calcitonin gene-related peptide (CGRP) receptor antagonist, CGRP_(8-37), completely blocked the nicotine-induced dilation. In both arteries, the vasodilation was also significantly decreased by glibenclamide, an ATP-sensitive K⁺ (K⁺_ATP) channel inhibitor. Levcromakalim, a K⁺_ATP channel opener, caused a dilation that was blocked by glibenclamide in both arteries. In the pulmonary artery, NO donor-mediated dilation was significantly decreased by pre-treatment with glibenclamide. The physiological data were supported by NADPH-diaphorase histochemistry and immunohistochemistry, which demonstrated NOS in perivascular nerve fibres but not the endothelium of the arteries. These results indicate that the pulmonary and cutaneous arteries of B. marinus are regulated by NO from nitrergic nerves rather than NO released from the endothelium. The nitrergic vasodilation in the arteries appears to be caused, in part, via activation of K⁺_ATP channels. Thus, NO could play an important role in determining pulmo-cutaneous blood flow and the magnitude of cardiac shunting.