A novel broad host range antimicrobial substance, Thermophilin 13, has been isolated and purified from the growth medium of Streptococcus thermophilus. Thermophilin 13 is composed of the antibacterial peptide ThmA (Mr of 5776) and the enhancing factor ThmB (Mr of 3910); the latter peptide increased the activity of ThmA approximately 40 x. Both peptides are encoded by a single operon, and an equimolar ratio was optimal for Thermophilin 13 activity. Despite the antilisterial activity of Thermophilin 13, neither ThmA nor ThmB contain the YGNGV-C consensus sequence of Listeria-active peptides, and post-translational modifications comparable to that in the lantibiotics are also absent. Mass spectrometry did reveal the apparent oxidation of methionines in ThmA, which resulted in a peptide that could not be enhanced any longer by ThmB, whereas the intrinsic bactericidal activity was normal. Thermophilin 13 dissipated the membrane potential and the pH gradient in liposomes, and this activity was independent of membrane components from a sensitive strain (e.g. lipid or proteinaceous receptor). Models of possible poration complexes formed are proposed on the basis of sequence comparisons, structure predictions, and the functional analysis of Thermophilin 13.