The tridecapeptides Asn(13)-orcokinin and Val(13)-orcokinin, two known members of the orcokinin neuropeptide family native to crustaceans, and a novel octapeptide, orcomyotropin, FDAFTTGFamide, have been identified from extracts of hindguts of the crayfish Orconectes limosus using an isolated hindgut contractility bioassay, high-performance liquid chromatography, microsequencing and mass spectrometry. All three peptides display strong inotropic actions on crayfish hindguts. Orcomyotropin showed higher potency than the two orcokinins. Threshold concentration was approximately 5 x 10(-12)mol l(-1)versus 10(-10)mol l(-1) for the two orcokinins. An approximately fivefold increase in contraction amplitude was observed with 10(-9)mol l(-1) orcomyotropin and 10(-7)mol l(-1) of the orcokinins. Asn(13)- and Val(13)-orcokinin did not differ significantly with regard to their biological effects. Semi-isolated crayfish hearts and locust oviducts did not respond to the three peptides. Immunocytochemistry using antisera against Asn(13)-orcokinin and orcomyotropin showed that these neuropeptides are co-localized in approximately 80-90 neurones of the terminal abdominal ganglion that have been shown to innervate the entire hindgut muscularis via the intestinal nerve. The neurones form elaborate terminal branches preferentially on longitudinal hindgut muscles. Orcomyotropin is a novel crustacean member of the GF-amide family of myotropic and/or allatotropic neuropeptides from annelids, molluscs and insects.