Leuconostoc MF215B was found to produce a two-peptide bacteriocin referred to as leucocin H. The two peptides were termed leucocin H alpha and leucocin H beta. When acting together, they inhibit, among others, Listeria monocytogenes, Bacillus cereus, and Clostridium perfringens. Production of leucocin H in growth medium takes place at temperatures down to 6 degrees C and at pH below 7. The highest activity of leucocin H in growth medium was demonstrated in the late exponential growth phase. The bacteriocin was purified by precipitation with ammonium sulfate, ion-exchange (SP Sepharose) and reverse phase chromatography. Upon purification, specific activity increased 10(5)-fold, and the final specific activity was 2 X 10(7) BU/OD280. Amino acid composition analyses of leucocin H alpha and leucocin H beta indicated that both peptides consisted of around 40 amino acid residues. Their N-termini were blocked for Edman degradation, and the methionin residues of leucocin H beta did not respond to Cyanogen Bromide (CNBr) cleavage. Absorbance at 280 nm indicated the presence of tryptophan residues and tryptophan-fracturing opened for partial sequencing by Edman degradation. From leucocin H alpha, the sequence of 20 amino acids was obtained; from leucocin H beta the sequence of 28 amino acid residues was obtained. No sequence homology to other known bacteriocins could be demonstrated. It also appeared that the two peptides themselves shared little or no sequence homology. The presence of soy oil did not affect the activity of leucocin H in agar.