The distribution of the autonomic nervous system in the rat seminal vesicle was studied with immuno- and enzyme-histochemistry. In the smooth muscle layer, a large number of neuropeptide Y (NPY)- and tyrosine hydroxylase (TH)-immunoreactive nerve fibers were observed, while few fibers were distributed in the mucosal layer. A small number of substance P-immunoreactive fibers were present only in the smooth muscular layer, but vasoactive intestinal polypeptide (VIP)-immunoreactive fibers were abundantly distributed in both layers. In the mucosal layer, the VIP-fibers ran attached to blood vessels and encircled the glandular cavities. Acetylcholinesterasepositive fibers were also observed in the mucosal and muscular layers. Electron-microscopic studies revealed that NPY- and TH-containing nerve terminals were in apposition to smooth muscle cells, and VIP-fibers terminated at blood vessels. These results suggest different modes of action of NPY-, TH- and VIP-containing nerve fibers in the seminal vesicle.