The acidic O‐specific polysaccharide chain (O‐antigen) of the lipopolysaccharide (LPS) of Proteus mirabilis strain D52 was studied using chemical analyses along with 1H‐NMR and 13C‐NMR spectroscopy, including 2D COSY, TOCSY, ROESY, H‐detected 1H,13C and 1H,31P HMQC experiments. The polysaccharide was found to contain d‐ribitol 5‐phosphate (d‐Rib‐ol‐5‐P) and ethanolamine phosphate (Etn‐P) and has the following structureThis structure is identical with that of the O‐polysaccharide of P. mirabilis O33 strain 59/57, and, hence, P. mirabilis D52 belongs to the same Proteus serogroup O33. Serological studies with O‐antiserum against P. mirabilis D52 confirmed this but showed that the LPS species of P. mirabilis 59/57 and D52 are not identical, having different epitopes in the core region. A serological cross‐reactivity of P. mirabilis D52 O‐antiserum was observed with LPS of two other Proteus strains, P. mirabilis O16 and P. penneri 103, which have structurally different O‐polysaccharides. The role of charged groups, Rib‐ol‐5‐P and Etn‐P in the immunospecificity is discussed.