The possible implication of nitric oxide synthase (NOS) in penile erection was examined by utilizing NADPH histochemistry in the rat. NADPH histochemistry indicated that the major pelvic ganglion (MPG), a well-known origin of nerve fibers supplying the external genitalia, contained many NOS-positive neurons. On the other hand, NOS-positive nerve fibers in penile erectile tissue observed in the walls of both arteries and veins, as well as in intrinsic smooth muscles. The retrograde tracing study with Fluoro-Gold (FG) in combination with NADPH histochemistry revealed that almost all MPG neurons which were retrogradely labeled with FG injected into the penile crura were NOS-positive. Thus, the MPG was considered to be the main source of NOS-positive nerve fibers in penile erectile tissue.