Matrix-assisted laser desorption/ionization time-of-flight and tandem time-of-flight (MALDI-TOF and MALDI-TOF/TOF) mass spectrometry were used to sequence and localize three novel, related neuropeptides in the nervous system of the nematode Ascaris suum, AMRNALVRFamide (AF21), NGAPQPFVRFamide (AF22), and SGMRNALVRFamide (AF23). The amino acid sequences were used to clone a novel neuropeptide gene (afp-6) that encodes a precursor bearing a single copy of each of the peptides. In situ hybridization and immunocytochemistry revealed that both the transcript and the peptides are expressed in a single cell in the ventral ganglion. Pharmacological studies of intact nematodes injected with these peptides, as well as physiological studies of responses to them in muscle tissue, motor neurons, and the pharynx, reveal that these peptides have potent bioactivity in the locomotory and feeding systems. Further exploration of their effects may contribute to our understanding of neuropeptide modulation of behavior and also to the development of compounds with anthelmintic relevance. J. Comp. Neurol. 502:872–882, 2007. © 2007 Wiley-Liss, Inc.