The regional variation of nitric oxide synthase (NOS)-containing nerve fibres along the length of the vas deferens and cauda epididymidis was investigated in cross-sections of abdominal, intermediate and scrotal portions of the vas deferens as well as the cauda epididymidis of the rat. In adult rats, antibody to protein gene product 9.5 (protein found in all neurones) revealed very rich immunostaining in the muscle layers from all four areas studied. In the abdominal portion of the vas deferens NOS immunostaining revealed weak immunofluorescent nerve fibres just beneath the epithelium in the lamina propria extending into the innermost muscle layer where they became less dense. A similar pattern of NOS immunostaining was also seen in sections from the intermediate portion of the vas deferens. Fluorescent nerve fibres were not present in the outer muscle layers. Very few, if any, NOS-immunoreactive nerves were seen in scrotal portions of the vas deferens and cauda epididymidis. In tissue segments taken from aged rats the number of NOS-immunoreactive nerve fibres was increased in all segments, but were still more concentrated at the abdominal end of the male reproductive tract. In contrast, sections of vas deferens from immature rats showed no NOS immunostaining. These findings suggest that nitrergic nerves innervating the rat vas deferens may be involved in the initial relaxation of the abdominal portion of the vas deferens which allows for unidirectional sperm transport. The age-dependent increase in NOS-containing nerve fibres suggests that their development is also androgen-dependent.