A new bacteriocin produced by Bacillus thuringiensis subsp. entomocidus was identified. The antibacterial activity termed entomocin 110 was produced starting at mid-logarithmic growth phase, reaching its maximum at the early and during stationary phase. The bacteriocin obtained from culture supernatant was inhibitory to several Gram-positive bacteria including Listeria monocytogenes, Paenibacillus larvae and other Bacillus species. Entomocin 110 was shown to be heat stable and resistant to pH variation and to organic solvents. The inhibitory activity was totally lost after proteinase K treatment, thereby revealing its proteinaceous nature. The mode of action of entomocin 110 was bactericidal and bacteriolytic. Upon partial purification with ammonium sulphate precipitation followed by butanol extraction, an active peptide with an apparent molecular weight of 4.8 kDa was identified. Cross inhibition tests with bacteriocin producer strains and plasmid profiles indicated that entomocin 110 is a new bacteriocin, which genetic determinants are probably harbored by the chromosome.