Colicins are gram-negative bacteriocins produced by and effective against Escherichia coli and related species. Colicin E1 (ColE1) is composed of three functional domains, which collectively have a pore-forming effect on targeted bacteria. ColE1 binding and translocation domains are highly specific in contrast to the pore-forming domain, implying that ColE1 could be broadly effective. In this study, the activity of ColE1 against Listeria monocytogenes was evaluated in broth and on surfaces of ready-to-eat products. Individual strains of L. monocytogenes were examined in broth containing ColE1 at 0, 0.1, 1, or 10 μg/ml. Although strain differences in sensitivity to ColE1 existed, growth was significantly reduced in all strains at doses as low as 0.1 μg/ml. Sterilized ham slices were submerged in a five-strain L. monocytogenes cocktail (either 7 or 4 log CFU/ml) and placed in vacuum packages containing 0, 1, 5, 10, 25, or 50 μg of ColE1. Ham slices were then stored at 4 or 10°C, and samples were removed and examined for L. monocytogenes after 1, 3, 7, and 14 days. Reduction of L. monocytogenes by ColE1 was dependent on initial inoculum concentration and storage temperature. For slices stored at 4°C, treatment with 25 μg reduced Listeria growth below detection limits for the slices inoculated with 4 log CFU/ml for the entire 14 days, whereas for the 7-log CFU/ml slices, growth was detected at 7 days postinoculation. For slices stored at 10°C, 10 μg/ml ColE1 significantly inhibited growth of L. monocytogenes for up to 3 days for both inoculation groups. These data indicate that ColE1 is highly effective against Listeria.