To determine the proportion of nerve fibers in the hypogastric nerve (HGN) and pelvic splanchnic nerve (PSN), small tissue strips of the HGN and PSN from 12 donated elderly cadavers were examined histologically. Immunohistochemistry for neuronal nitric oxide synthase (NOS), vasoactive intestinal peptide (VIP), and tyrosine hydroxylase (TH) was performed. More than 70% of fibers per bundle in the HGN were positive for TH at the level of the sacral promontory. In addition, NOS- (negative) and/or VIP+ (positive) fibers were observed in small areas of each nerve bundle, although the proportion of each was usually less than 10%. In the PSN near the third sacral nerve root, the proportion of nerve fibers positive for NOS and/or VIP (or TH) was below 30%. In both the HGN and PSN, the number of VIP+ fibers was usually greater than that of NOS+ fibers, with frequent co-localization of NOS and VIP. More fibers in both nerves were positive for TH than for these other markers. In contrast to pelvic plexus branches, there were no differences in the proportions of NOS+ and VIP+ fibers between nerve bundles in each of the tissue strips. Thus, target-dependent sorting of nerve fibers was not apparent in the HGN at the level of the sacral promontory or in the PSN near the third sacral nerve root. The NOS+ and/or VIP+ fibers in the HGN were most likely ascending postganglionic fibers to the colon, while those in the PSN root may be preganglionic fibers from Onuf's nucleus.