Vasoactive intestinal polypeptide was localized by immunocytochemical techniques in the major pelvic ganglion and penile erectile tissue of the rat. Vasoactive intestinal polypeptide fibers were concentrated in penile crura with the density of innervation decreasing distally. The helicine arteries were very densely innervated while fewer fibers surrounded the deep artery of the penis. Intrinsic smooth muscle of the cavernous bodies received a moderate supply of vasoactive intestinal polypeptide immunoreactive fibers. Dorsal vascular structures, including the deep dorsal vein were innervated by vasoactive intestinal polypeptide fibers. Vasoactive intestinal polypeptide immunoreactive cell bodies were found in the major pelvic ganglion, concentrated on one end of the ganglion. Retrograde studies with a dye injected into the penile crura indicated that neurons in major pelvic ganglion projected to the penis. Combined dye and immunofluorescent studies showed that all the dye-labeled neurons were immunoreactive for vasoactive intestinal polypeptide. It is concluded that all vascular beds in the penis of the rat are innervated by vasoactive intestinal polypeptide fibers and that the extent of the innervation is related to the occurrence of smooth muscle. Neurons in the major pelvic ganglion probably are the main source of vasoactive intestinal polypeptide fibers to the penis.