Nitric oxide synthase (NOS)- and vasoactive intestinal peptide (VIP)-immunoreactive neurons projecting to the upper rectum or penis were examined using retrograde tracing combined with immunohistochemistry in the major pelvic ganglion of male rats. Five days after injection of Fluoro-Gold (FG) into the upper rectum or penis, the major pelvic ganglion was treated with colchicine. FG injected into the upper rectum labelled many ganglion neurons in the major pelvic ganglion. Immunohistochemistry showed that 37% of FG-labelled neurons were immunoreactive for NOS and 33% for VIP. After injection of FG into the penis, 41% of FG-labelled neurons were immunoreactive for NOS and 25% for VIP. Serial cryostat sections stained for NOS and VIP, respectively, showed the co-localization of NOS and VIP in the ganglion cells projecting to the rectum and penis. In the major pelvic ganglion of the colchicine-treated animals, about 17% of the ganglion cells were immunoreactive for NOS and 32% were immunoreactive for VIP. These neurons were small in diameter (less than 30 microns). A histogram showing cell sizes in cross-sectional areas of NOS-immunoreactive neurons coincided with that of VIP-immunoreactive neurons. Most of the NOS- and VIP-immunoreactive neurons were less than 600 microns. These results indicate that small neurons containing both NOS and VIP in the major pelvic ganglion project to the rectum and penis. In the penile erectile tissues and enteric ganglia, NO and VIP may be released from the same axons and may act concomitantly on the target tissue.