Lipid A from Rhodobacter capsulatus 37b4 consists of a d-glucosaminyl-(β1–6)-d-glucosamine disaccharide backbone, carrying diphosphorylethanolamine at C-1 of the reducing glucosamine and phosphorylethanolamine at C-4′ of the nonreducing glucosamine. 1,4′-Bisphosphorylated lipid A, lacking the polar head groups, was also encountered and contributed to the observed microheterogeneity in the phosphate substitution. The amino functions of both glucosamines are substituted almost entirely by the rare 3-oxotetradecanoic acid, which is a characteristic constituent of lipid A in the genus Rhodobacter. 3-Hydroxydecanoic acid is ester-bound at C-3 and C-3′ of the glucosamine disaccharide and the one at the nonreducing glucosamine (C-3′) is partially substituted by dodecenoic acid to form an ester-bound diester. In free lipid A, hydroxy groups at C-4 and C-6′ of the glucosamine disaccharide are unsubstituted, C-6′ being the putative attachment point of the lipopolysaccharide core. The nontoxic Rhodobacter capsulatus lipid A shows extensive serological cross-reaction with the toxic Salmonella lipid A. Structural similarities in the hydrophilic part of both types of lipid A, dissimilarities in the hydrophobic part and their impacts on serologic properties are discussed.