Using a radioimmunoassay for the carboxyl-terminal sequence Arg-Val-NH₂, two novel peptides were purified from extracts of the sea anemone Anthopleura elegantissima. These peptides were L-3-phenyllactyl-Tyr-Arg-Ile-NH₂ (name: Antho-RIamide I) and its des-phenyllactyl fragment Tyr-Arg-Ile-NH₂ (Antho-RIamide II). Immunocytochemical staining showed that these peptides were localized in neurons of sea anemones. Application of low concentrations (10^-8 M) of Antho-RIamide I inhibited spontaneous contractions in several muscle groups of sea anemones, whereas Antho-RIamide II was inactive. Antho-RIamide I is the second neuropeptide from sea anemones that bears the unusual, amino-terminal L-3-phenyllactyl blocking group. We suggest that this group renders the peptide resistant against degradation by nonspecific aminopeptidases. In addition, the L-3-phenyllactyl residue might also play a role in receptor binding.