Male copulation behavior in mollusks is controlled by an array of peptide messengers. In the present study, we have used a peptidomics approach employing liquid chromatography in conjunction with electrospray mass spectrometry to characterize peptides contained in the penial complex of the freshwater snail, Lymnaea stagnalis. In addition to the previously described peptides, we have identified a group of novel peptides that share the carboxyl termini of -FVRlamide. A cDNA cloning study revealed the organization of the precursor, which contains 20 peptide domains with the carboxyl termini of -F(X)Rlamide which are flanked by many putative proteolytic sites including the KR and the less commonly occurring (G)K and (G)R sites. In addition, there are several monobasic R and dibasic RR and KK sites that may be used for processing. We then used MALDI-TOF/TOF-MS in a data-dependent mode, which selected all the molecular ion species with the predicted masses of the mature -F(X)-Rlamide peptides, and performed MS/MS analysis on these peptides. This approach allowed us to identify all the predicted -F(X)Rlamide peptides. Immunocytochemistry showed the localization of -FVRlamide immunoreactive neurons in several central ganglia, and immunoreactive axons in the penial complex. Finally, application of synthetic -FVRlamide peptides to an in vitro posterior vas deferens preparation showed inhibitory effect on the spontaneous contraction/relaxation cycle of the vas deferens. © 2006 American Chemical Society.