Specimens of penile erectile tissue from the corpus cavernosum (CC) and corpus spongiosum (CS) of beagle dogs were investigated with reference to morphological and functional aspects of the nitric oxide (NO) system. Using immunohistochemistry, the smooth muscle bundles of the CC were found to contain a rich innervation, as seen by the pan-neuronal marker, protein gene product 9.5. A large number of nerves also stained positively for acetylcholine esterase. There was a moderate to rich supply of nerves containing NO synthase, which was frequently co-localized with vasoactive intestinal polypeptide. CS preparations from the proximal penis (the spongious bulb) had an innervation similar to that of the CC. However, CS preparations from the distal penis had a distinctly lower density of nerves than the proximal part, and spongious tissue from the glans penis was practically devoid of nerves. In isolated preparations of proximal and distal CC and CS, contracted by noradrenaline, electrical field stimulation (EFS) produced frequency-dependent relaxations. The responses were less pronounced in preparations from the distal penis, particularly in the CS. Pre-treatment with the NO-synthesis inhibitor NG-nitro-L-arginine abolished all relaxations. Carbachol effectively relaxed noradrenaline-contracted strips. The responses were similar in proximal preparations from the CC and CS, and significantly less pronounced in strips from the distal penis. NG-nitro-L-arginine significantly reduced the relaxant effect of carbachol. Preparations of spongious tissue from the glans penis did not react to high potassium solution, addition of noradrenaline, or EFS. The results demonstrate regional differences within the CS, both in innervation and functional responses.