Fourteen Lactococcus lactis strains showing inhibitory activity against Listeria innocua SICC 4202 were isolated from different French raw milks and raw milk cheeses and screened for bacteriocin production by the triple layer method under conditions that eliminate the effects of lactic acid and hydrogen peroxide. Three bacteriocinogenic strains (two Lactococcus lactis subsp. lactis bv. diacetylactis UL719 and UL720 and one Lactococcus lactis subsp. lactis UL730) were selected for their high capacity to inhibit the growth of various food pathogens, including Listeria monocytogenes, Staphylococcus aureus, and clostridial strains. The inhibitory compounds from these three strains are inactivated by selected proteases, indicating their protein nature. They retained their antibacterial activity after heat treatments of 100 degrees C for 60 min and 121 degrees C for 20 min, and in the pH range from 2 to 11. The bacteriocin diacetin B produced by strain UL720 has been purified by a pH-dependent adsorption-desorption procedure, followed by reverse-phase high performance liquid chromatography, with a yield of 1.25% of the original activity. Mass spectrometry analysis indicates that the pure peptide has a molecular mass of 4292.32 or 4490.28 Da, while amino acid sequencing allowed the identification of the primary structure of the bacteriocin composed of 37 amino acid residues. The structure of the peptide did not show similarity with other known bacteriocins from lactic acid bacteria.