Retrograde dye staining, enkephalin immunocytochemistry and nerve lesion paradigms were used to determine if penile neurons in the pelvic plexus are innervated by fibers in the hypogastric nerve. In the intact major pelvic ganglion of the rat, some 80% of penile neurons are enclosed by an enkephalin-positive fiber plexus. Following surgical interruption of the pelvic nerve, 20% of penile neurons were still surrounded by an enkephalin plexus. After interruption of the pelvic nerve and the hypogastric nerve, the enkephalin plexus in the ganglion was virtually absent, including the plexus around penile neurons. Therefore, possible intrinsic sources of the enkephalin fibers such as enkephalin-positive principal neurons and small intensely fluorescent cells, do not account for the delicate enkephalin fiber system in the pelvic ganglion. It is concluded that the pelvic nerve is the major source of preganglionic innervation to penile neurons in the major pelvic ganglion. However, it is significant that the hypogastric nerve is preganglionic to about 20% of penile neurons. The pathway through the hypogastric nerve may represent an alternate vasodilator system to penile erectile tissue.