In 15 adult dogs, the possible role of acetylcholine as a parasympathetic neurotransmitter in canine penile erection was investigated. Intracavernous injection of increasing dosages of acetylcholine (0.1–100 µg) induced a dose-dependent erectile response with increased arterial flow, cavernous smooth muscle relaxation, and venous occlusion. This erectile response was completely abolished after muscarinic blockade by intracavernous injection of 0.1 mg atropine. After cavernous nerve stimulation, atropine injection significantly reduced the pudendal arterial flow (by 25%) and likewise caused a significant reduction in cavernous outflow restriction. Histologic staining showed acetylcholinesterase-positive fibers around the cavernous arteries and within the cavernous erectile tissue.