Two new polypeptide components which exhibited an analgesic effect in experiments on mice were isolated from the Heteractis crispa sea tropical anemone by the combination of chromatographic methods. The APHC2 and APHC3 new polypeptides consisted of 56 amino acid residues and contained six cysteine residues. Their complete amino acid sequence was determined by the methods of Edman sequencing, mass spectrometry, and peptide mapping. An analysis of the primary structure of the new peptides allowed for their attribution to a large group of trypsin inhibitors of the Kunitz type. An interesting biological function of the new polypeptides was their analgesic effect on mammals, which is possibly realized via the modulation of the activity of the TRPV1 receptor and was not associated with the residual inhibiting activity towards trypsin and chymotrypsin. The analgesic activity of the APHC3 polypeptide was measured on the hot plate model of acute pain and was significantly higher than that of APHC2. Methods of preparation of the recombinant analogues were created for both polypeptides. © 2009 Pleiades Publishing, Ltd.