Nitric oxide synthase (NOS), which catalyzes the production of nitric oxide (NO), was characterized within the reproductive tract of adult male Sprague-Dawley rats by means of biochemical and immunohistochemical techniques. Tissues examined included the testis, epididymis (caput, corpus, and cauda regions), vas deferens, ejaculatory duct, seminal vesicle, and coagulating gland. NOS activity was measured by use of an assay based on the stoichiometric conversion of [3H]-L-arginine to [3H]-L-citrulline and NO, catalyzed by NOS. Low levels of NOS activity were detected in the testis and seminal vesicle (< 0.5 fmol [3H]-L-citrulline formed/min/mg protein in each tissue). The highest levels of NOS activity were present in the cauda segment of the epididymis and in the vas deferens, each having a sevenfold greater amount of NOS activity than the testis (p < 0.05). Intermediate levels of NOS activity were detected in the coagulating gland (0.863 +/- 0.248 fmol [3H]-L-citrulline formed/min/mg protein), caput epididymidis (0.457 +/- 0.180 fmol [3H]-L-citrulline formed/min/mg protein), and corpus epididymidis (0.631 +/- 0.215 fmol [3H]-L-citrulline formed/min/mg protein). NADPH diaphorase histochemistry and NOS immunohistochemistry localized NOS to neuronal fibers coursing throughout the smooth musculature and subepithelial regions of the epididymis, vas deferens, and ejaculatory duct. Endothelial cells and nerve plexuses within the adventitia of blood vessels supplying reproductive tissues were also positive for NOS. Additional localizations of NOS were within epithelial cells of the epididymis and coagulating gland.(ABSTRACT TRUNCATED AT 250 WORDS)