The cholesterol-dependent cytolysins (CDCs) are pore-forming toxins that have been exclusively associated with a wide variety of bacterial pathogens and opportunistic pathogens from theFirmicutes and Actinobacteria, which exhibit a Gram-positive type of cell structure. We have characterized the first CDCs from Gram-negative bacterial species, which include Desulfobulbus propionicus type species Widdel 1981 (DSM 2032)(desulfolysin [DLY]) and Enterobacter lignolyticu(formerly Enterobacter cloacae) SCF1 (enterolysin [ELY]). The DLY and ELY primary structuresshow that they maintain the signature motifs of the CDCs but lack an obvious secretion signal. Recombinant, purified DLY (rDLY) and ELY (rELY) exhibited cholesterol-dependent binding and cytolytic activity and formed the typical large CDC membrane oligomeric pore complex. Unlike the CDCsfrom Gram-positive species, which are human- and animal-opportunistic pathogens, neither D.propionicus nor E. lignolyticus is known to be a pathogen or commensal of humans or animals: thehabitats of both organisms appear to be restricted to anaerobic soils and/or sediments. Thesestudies reveal for the first time that the genes for functional CDCs are present in bacterial species that exhibit a Gram-negative cell structure. These are also the first bacterial species containing a CDC gene that are not known to inhabit or cause disease in humans or animals, which suggests a role of these CDCs in the defense against eukaryote bacterial predators.