Using enzyme-linked immunosorbent assays, a dot-immunobinding assay and a three-step reverse-phase HPLC separation, four Arg-Phe-amide (RFamide) peptides were purified from sex segmental ganglia extracts of the leech Erpobdella octoculata; FMRFamide, FM(O)RFamide, FLRFamide and GDPFLRFamide. Their amino acid sequences were elucidated by means of a combined approach using antiserum specificity, synthetic-peptide coelution, automated Edman degradation and electrospray mass spectrometry. One of these peptides, GDPFLRFamide, is a novel leech RFamide neuropeptide. Two of the above RFamide peptides are involved in the control of leech hydric balance; one (GDPFLRFamide) is diuretic, the other (FMRFamide) is anti-diuretic. Titration of each purified RFamide peptide indicated a similar amount of each tetrapeptide and of tetrapeptides and heptapeptides. A comparison between RFamide peptides of E. octoculata and molluscs reveals structural similarities supporting the hypothesis for the existence of an ancestral RFamide peptide gene common to leeches and molluscs.