The O-specific polysaccharide of Proteus penneri strain 34 was studied using 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, NOESY, and H-detected 1H, 13C HMQC experiments. The following structure was established, which is unique among the known structures of Proteus O-antigens:-->4)-beta-D-Glcp-(1-->3)-beta-D-GalpNAc-(1-->4)-beta- D-GalpNAc-(1-->4)-beta-D-Galp-(1-->. Accordingly, no cross-reaction was observed between P. penneri 34 O-antiserum and O-antigens of other Proteus strains. Therefore, the strain studied should belong to a new Proteus serogroup O65.