Immunohistochemical studies of the vas deferens and seminal vesicle of mouse, guinea-pig, and rabbit showed the presence of nerve fibres containing vasoactive intestinal polypeptide (VIP), substance P (SP), and gastrin-releasing peptide (GRP) supplying the smooth muscle layers as well as blood vessels. The nerve supply was better developed in the seminal vesicle than in the vas deferens. The motor activity of the vas deferens and seminal vesicle of the guinea-pig was studied in vitro. The vas deferens responded to transmural electrical stimulation with a twitch followed by a slow contraction. The twitch was blocked by guanethidine and tetrodotoxin, but not by atropine, propranolol, phenoxybenzamine, or fluphenazine. The slow contraction exhibited features of an alpha-receptor-mediated response. SP, physalaemin and eledoisin contracted the smooth muscle and also potentiated the twitch response to electrical nerve stimulation in a concentration-dependent manner. The SP blocking agent, (D-Pro2,D-Trp7,9)-SP, affected neither the resting tension nor the response to electrical stimulation. It is therefore suggested that the SP fibres act mainly prejunctionally. VIP, Leu-enkephalin, cholecystokinin octapeptide (CCK-8), angiotensin II, vasopressin, neurotensin, bombesin, and GRP had no effect on either the resting tension or the response to electrical nerve stimulation. The seminal vesicle responded to electrical stimulation with a contraction which was unimpaired by atropine, propranolol, phenoxybenzamine, and guanethidine, but abolished by tetrodotoxin. Hence, this contraction is mediated by a non-adrenergic, non-cholinergic neurotransmitter. Bombesin, GRP, SP, physalaemin and eledoisin contracted the smooth muscle and potentiated the response to electrical stimulation. VIP, Leu-enkephalin, CCK-8, angiotensin II, vasopressin, and neurotensin had no effect on the resting tension or on the response to transmural electrical stimulation. The SP antagonist abolished the contraction elicited by SP but did not influence the response to nerve stimulation. The results suggest that the SP and GRP nerves may have prejunctional and facilitating postjunctional effects in the seminal vesicle.