The biological effects of neuropeptide Y (NPY), rat pancreatic polypeptide (rPP), hybrid analogs of NPY and PP, and C-terminal fragments of NPY were studied in the field-stimulated rat vas deferens model. The results were correlated with peptide binding experiments in Y1 and PP receptor assays on rat PC-12 cells and Y2 receptors on porcine hippocampal membranes. NPY and rPP inhibited the electrically induced contractions in the vas deferens with an IC50 of 25 and 22 nM respectively. However, in contrast to NPY, rPP could not totally block muscle activity. The inhibitory action of the long C-terminal fragment of NPY, NPY-(19-36) and NPY-(11-36), indicated that NPY acts through a Y2 receptor in the vas deferens. The structural basis for the differential recognition of NPY and PP by Y2 receptors and partly also by PP receptors, could be defined with hybrid analogs of PP and NPY. The analogs, [Ile31,Gln34]PP and [Leu31,Pro33]NPY reacted in the vas deferens preparation in accordance with their relative potency in the Y2 and PP receptor assays. [Ile31,Gln34]PP, which bound to the Y2 receptor like NPY, was also able to block the part of the contractile response which was resistant to rPP. It is concluded that in the vas deferens, PP-fold peptides act through two types of receptors: Y2 and PP, and that residues in the C-terminal part of the molecules determine the differential recognition of the peptides by these receptor types.