Analogues of KNEFIRFamide (Lys-Asn-Glu-Phe-Ile-Arg-Phe-NH2; AF1), an FMRFamide-related peptide (FaRP) originally isolated from Ascaris suum, were characterized in an A. suum muscle tension assay. AF1 had biphasic effects on this preparation, inducing a brief relaxation followed by excitation and spastic paralysis. Activity of AF1 in this assay was eliminated by N-terminal deletions and by deamidation of the carboxy-terminus. The potency of AF1 was greatly reduced by alanine substitution for any residue. Peptides that retained activity did not show the biphasic response observed with AF1, suggesting that the inhibitory and excitatory phases seen with AF1 may be due to activation of distinct receptors. The basis for the marked differences in potency observed between AF1 and the structurally related nematode FaRP, AF2 (KHEYLRFamide) was also tested. AF2 is approximately 1000-fold more potent than AF1 in this assay, but has physiological effects that are otherwise indistinguishable. KNEYIRFamide and KNEFLRFamide induced characteristic AF1/AF2 responses, but were much less potent than the native peptides. In contrast, KHEYIRFamide resembled AF1 in potency and pattern of responses. These data suggest that AF1 and AF2 act at distinct receptors, and hypothesis supported by the observation that KNEFIAFamide antagonized the effects of AF1 but not of AF2.