A wall-surface protein antigen, designated 32K antigen, was extracted from whole cells of Clostridium difficile strain ATCC 11011 with phosphate buffered saline and purified by ion-exchange chromatography, gel filtration, and chromatofocusing. The 32K antigen preparation was determined to be highly homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid composition of the antigen was characteristic in the predominance of the acidic amino acids, the very low contents of methionine and histidine, and the lack of cysteine. A monomeric molecular weight of the 32K antigen was estimated to be 32,000 by SDS-PAGE and 30,200 by sedimentation equilibrium. The antigen exhibited two isoelectric forms (IP, 4.12 and 3.96). Neither carbohydrate nor phosphorus was detectable in the antigen. The antigen was relatively resistant to trypsin but sensitive to pepsin. Immunoblot analysis of the wall proteins isolated from other strains of C. difficile probed with monospecific antiserum against the antigen from ATCC 11011 showed that the antigenicity of 32K wall protein was common among some of the strains containing 32K wall proteins.