A multimolecular complex of polypeptides found associated with the lipopolysaccharide endotoxin in Salmonella, referred to as endotoxin-associated protein (EP), has been extracted from a rough strain of Salmonella typhimurium which does not synthesize 0 antigens. Since standard methods of extraction applicable to smooth strains of Salmonella were not successful for this rough strain, two modified procedures were developed. The resulting products were similar to smooth EP in terms of their biochemical, physical and mitogenic properties. When the immunogenicity of the rough EP was characterized by a protection assay in mice challenged with virulent Salmonella, it was found that the rough EP preparations were protective; however, they were not as active as the EP from a smooth strain of S. typhimurium.