Clostridium difficile is a major cause of pseudomembranous colitis following antimicrobial therapy. There is evidence to suggest that this organism may be hospital acquired. Polyacrylamide gel electrophoretic (PAGE) analysis of protein profiles of C. difficile cell extracts was examined for possible usefulness in epidemiologic studies. At least 50 bands could be distinguished in soluble cell extracts of C. difficile. Freezethawing of extracts and/or length of storage time did not affect the protein profiles. While all strains tested were nearly identical, several strains were unique in their lack of a 34,000-dalton polypeptide. Protein patterns of C. difficile could easily be distinguished from those of Clostridium sordellii. Additionally, surface antigens extracted from several strains of C. difficile and from a few strains of C. sordellii revealed marked differences. Ethylenediaminetetraacetate (EDTA) extracts from C. difficile showed two or three major bands in PAGE analysis; strains could be divided into two major subgroups on the basis of band distribution. EDTA extracts from C. sordellii bore no similarity to those of C. difficile.