A novel antimicrobial peptide was isolated and characterized from the earthworm, Lumbricus rubellus. The antimicrobial peptide was purified to homogeneity by a heparin-affinity column and C18 reverse-phase HPLC, and named lumbricin I. Lumbricin I was a proline-rich antimicrobial peptide of 62 amino acids (15% proline in molar ratio; molecular mass, 7231 Da), whose complete sequence was determined by a combination of peptide sequence and cDNA analysis. The peptide and cDNA sequence analysis revealed that lumbricin I was produced as a precursor form consisting of 76 amino acids, with 14 residues in a presegment and 62 residues in mature lumbricin I. Lumbricin I showed antimicrobial activity in vitro against a broad spectrum of microorganisms without hemolytic activity. In addition, a 29-amino acid peptide, named lumbricin I(6-34), which was derived from residues 6-34 of lumbricin I, showed marginally stronger antimicrobial activity than lumbricin I. Northern blot analysis on total RNA revealed that expression of lumbricin I gene was not induced by bacterial infection, but was constitutively expressed. Furthermore, the expression of lumbricin I gene was specific in adult L. rubellus: Lumbricin I mRNA was detected only in adult L. rubellus, but not in eggs and young L. rubellus.