Five neuropeptides were isolated from CNS extracts of the horseshoe crab Limulus polyphemus by high pressure liquid chromatography (HPLC). The pep- tides were identified by radioimmunoassays (RIAs) based on two antisera raised to FMRFamide-related peptides (FaRPs). The purified peptides were analyzed by auto- mated sequencing and mass spectrometry, and the fol- lowing sequences were obtained: DEGHKMLYFamide, GHSLLHFamide, PDHHMMYFamide, DHGNMLY- Famide, and GGRSPSLRLRFamide. The first four pep- tides are members of a novel family with virtually no relationship to FMRFamide. GGRSPSLRLRFamide, on the basis of structural similarity, becomes the second member of a class of FaRPs known previously only from a peptide isolated from mosquito heads. At least one member of the novel family (GHSLLHFamide) inhibits the isolated heart of Limulus.