Substance P immunoreactivity in the major pelvic ganglion (MPG) of the rat was studied to define a possible role for this neuropeptide in functions of the pelvic portion of the autonomic nervous system. Substance P immunoreactivity was found in three locations in the ganglion: 1) as a plexus of varicose fibers, 2) in small intensely fluorescent (SIF) cells, and 3) after colchicine pretreatment, in some principal neurons. The perineuronal plexus of fibers appeared as small varicosities closely related to the somae of principal neurons. Approximately 10-20% of principal neurons were enclosed by a substance P-positive plexus. SIF cells were intensely stained for substance P. The general relationships of SIF cells in this ganglion were confirmed by their staining for substance P: their occurrence singly or in large clusters, their short tapering processes often related to principal neurons, and the occasional presence of a beaded process. Colchicine treatment resulted in the appearance of rare principal neurons that stained for substance P. The pelvic nerve was surgically interrupted to determine whether the perineuronal plexus of varicose fibers had an intrinsic origin or arose from cell bodies outside the ganglion. The perineuronal plexus was virtually absent following this procedure. The results of this study indicate that principal neurons in the major pelvic ganglion may be subject to the influence of substance P derived from two sources: 1) intrinsic substance P-containing SIF cells and 2) neurons probably residing in dorsal root ganglia. The nature of principal neurons that acquire staining for substance P after colchicine is unclear.