A complement-dependent bactericidal factor (RaRF) specific for the Ra chemotype strains of Salmonella and for the Ra-like strains of other enterobacteria has been found in sera of mice. We show here that the anti-Ra bactericidal factors are present in sera of all species, so far tested, of mammals, birds, reptiles, amphibians, and bony and cartilaginous fish. Certain properties, such as binding specificity, requirement of divalent cations for binding, and sensitivities to heat and reducing agents, of the factor in the representative animals were the same as or similar to those of the mouse RaRF. These results indicate that these factors have been conserved by vertebrates for more than 300 million years as a result of the necessity for the resistance to rough mutants of Gram-negative bacteria.