Rabbit polyclonal antibodies against the rough mutant lipopolysaccharide (LPS) of Salmonella minnesota R4 (chemotype Rd2P-) were serologically characterized by using R4 LPS, deacylated LPS, dephosphorylated LPS, and synthetic partial structures, including compounds comprising the core region of Rd2P- LPS bound to the beta 1-->6-linked glucosamine disaccharide with two amide-linked 3-hydroxytetradecanoic acid residues or coupled to bovine serum albumin. By using a passive hemolysis assay and an enzyme immunoassay and absorption and inhibition experiments, the antibody specificities present could be determined. One group of antibodies required components of the core oligosaccharide (with or without the side chain 3-deoxy-D-manno-octulosonic acid [Kdo]) and the phosphorylated glucosamine disaccharide of the lipid A moiety for binding. The phosphate-independent antibodies were directed against the core oligosaccharide, recognizing an epitope consisting of one terminal heptose linked to Kdo or to the reducing moiety of the alpha 2-->4-linked Kdo disaccharide. Antibodies requiring the presence of acyl residues and those reacting with a single heptose or Kdo residue were not detected.