A radioimmunoassay has been developed for peptides containing the carboxyl-terminal sequence Arg-Phe-NH2 (RFamide). Using this radioimmunoassay and applying cation-exchange chromatography and HPLC, we have isolated an RFamide peptide from acetic acid extracts of the sea anemone Anthopleura elegantissima. Three different methods established that the structure of the Anthopleura RFamide peptide (Antho-RFamide) is pyroGlu-Gly-Arg-Phe-NH2. Comparison of synthetic and natural Antho-RFamide and their enzymatic breakdown products on six different HPLC columns confirmed the structure of the sea anemone peptide. Using synthetic Antho-RFamide as a standard in our radioimmunoassay, we measured high concentrations (3.2 nmol/g wet weight) of this peptide in extracts of Anthopleura. It is proposed that Antho-RFamide is a transmitter at neuromuscular synapses in sea anemones.