This study was carried out to characterize the antilisterial substances produced by Leuconostoc sp. W65 and to evaluate the effects of pH, temperature, and time on inhibitory activity using response surface methodology. Leucocin W65, an antilisterial substance produced by Leuconostoc sp. W65, markedly inhibited the growth of Listeria monocytogenes, L. innocua, and L. ivanovii, whereas other pathogens including Gram-negative bacteria were not susceptible. The pH was the most effective factor with regard to bacteriocin activity, while temperature and time of heat treatment had no significant effect. Fifty percent of inhibitory activity remained after 22.8 min at pH 4.2 and $121^{circ}C$. Leucocin W65 was purified by ammonium sulfate precipitation, hydrophobic interaction chromatography, and tricine-SDS-PAGE. Compositional analysis originally estimated the peptide to be 56 amino acids in length without asparagine, glutamine, and tryptophane. The sequence of partial N-terminal amino acid residues of purified bacteriocin was identified as follows: $NH_{2}-XGXAGVXPXGGQQPXVPLXYP$.