Several outbreaks of Escherichia coli O157:H7 infections have been associated with contaminated alfalfa seeds. A recently isolated E. coli strain Hu194 was capable of inhibiting 22 strains of E. coli O157:H7 and this inhibition was mediated by the production of a colicin named Hu194. The objectives of this study were to test the efficacy of treating alfalfa seeds with colicin Hu194 against E. coli O157:H7 strains, and to characterize this antimicrobial protein. Significant reductions (approximately 5 log CFU ml-1) in the viable cell counts of strains 43890 and 43895 were observed after 1-day incubation with semi-crude colicin, and after 2 days for strain 3081. Strain 43890 was successfully eliminated (5 log CFU g-1) from inoculated alfalfa seeds after soaking in a colicin suspension at a concentration of 10,000 AU/g. Treatment of alfalfa seeds inoculated with strains 43895 and 3081 required 20-fold higher concentrations of colicin Hu194 to achieve as much as 3 log CFU g-1 reductions. The genes encoding the colicin Hu194 operon were located on a 6 kb plasmid, and the sequence analysis revealed that this colicin was an E-type DNAse. From the sequence data, the estimated molecular masses of colicin Hu194, its immunity protein and lysis protein were 61.3, 10.0 and 4.8 kDa, respectively. Based on DNA and protein sequence comparisons with other E-type colicin, colicin Hu194 belonged to the type E2-colicin cluster. However, cross-immunity tests between E-group colicins suggested that Hu194 colicin was divergent from the previously characterized E2 colicins.