We report the isolation of a novel pardaxin isoform from the toxic secretion of the Red Sea Moses sole (Pardachirus marmoratus). Mass spectrometrical analysis of the newly purified peptide revealed a different primary structure compared to the previously known pardaxin isoforms. Sequence analysis disclosed an aspartic acid residue instead of glycine at position 31 of the new isoform. According to the novel sequence, a synthetic Asp-31-peptide was compared with the native compound as well as with synthetic Gly-31-pardaxin. The isolated Asp-31-pardaxin isoform and its synthetic analog exhibited identical elution properties during reverse-phase HPLC, as well as similar dose-dependent lytic effects on human erythrocytes at a concentration of 10(-6) to 10(-5)M. The hemolytic activity of Asp-31-pardaxins was lower than that of Gly-31-pardaxin and no synergistic effect between these peptides was found. The additional negative charge introduced by Asp-31 is likely to affect the selectivity of pardaxin pores towards a variety of ions.