The purpose of the present investigation was to ascertain the functional significance of the reduction in cyclic AMP (cAMP) levels in the inhibitory action of neuropeptide Y (NPY) on [3H]noradrenaline ([3H]NA) release, as well as to further characterize the subtype(s) of NPY receptors involved in the peptide's actions in the rat vas deferens. We studied the effects of NPY, carboxyterminal fragments of this peptide and the NPY analog (Leu31,Pro34)-NPY on three functional responses, namely, the release of [3H]NA and the associated muscle contractions evoked by electrical stimulation, and the accumulation of cAMP stimulated by forskolin. NPY, a known inhibitor of the electrically-evoked [3H]NA release and neurogenic contractions is also a potent inhibitor of the forskolin-stimulated cAMP synthesis in the prostatic portion of the rat vas deferens. However, the ability of NPY to inhibit cAMP accumulation is lost upon tissue denervation, suggesting that this is likely to be a prejunctional effect. Elevation of cAMP levels by the use of the cell permeant analog of cAMP, 8-(p-chlorophenylthio)-cAMP (8pCPTcAMP) increases the electrically-evoked release of [3H]NA. However, the inhibition of [3H]NA release by NPY is not prevented by 8pCPTcAMP. Structure-activity relationship studies reveal that NPY and related peptides inhibit the release of [3H]NA, the muscle contractions and the synthesis of cAMP with a similar pharmacological profile. NPY is the most potent inhibitory agent, whereas [Leu31,Pro34]-NPY and NPY13-36, the respective Y1 and Y2 selective agonists, display similar potencies to inhibit the three responses. It is concluded that NPY inhibits neurotransmission in the rat vas deferens through the activation of a peptide receptor different from the known NPY-Y1 or NPY-Y2 receptor subtypes. NPY receptor activation in the vas deferens is negatively coupled to adenylyl cyclase activity. This intracellular signalling pathway is, however, not likely to mediate the peptide effects on the prejunctional regulation of noradrenaline release.