Colicin V is a ribosomally synthesized antimicrobial peptide produced by Escherichia coli. Four recently characterized genes, arranged in two convergent operons on the plasmid pCoIV-K30, are required for colicin V synthesis, export and immunity. We report the purification and N-terminal amino acid sequencing of the colicin V protein. Our results demonstrate that the colicin V primary translation product, which consists of 103 amino acids, is proteolytically processed. A leader peptide, consisting of 15 amino acid residues, is removed from the N-terminus during maturation of colicin V. This leader peptide is not related to the N-terminal signal sequences which direct proteins across the cytoplasmic membrane via the Sec pathway. The molecular mass of colicin V, obtained by mass spectrometry analysis, showed that the peptide consists of only unmodified amino acids. The deduced amino acid sequence of the leader peptide was highly homologous to the N-terminal extensions found in non-lantibiotic, peptide bacteriocins produced by Gram-positive bacteria. These findings strongly indicate that colicin V belongs to a family of small peptide bacteriocins that have been found previously only among the Gram-positive lactic acid bacteria.