The accurate and sensitive diagnosis of Clostridium difficile-related diarrhea, normally treated with vancomycin, has become increasingly important in light of the emergence of dangerous new strains of vancomycin-resistant enterococci. In order to improve the threshold for C. difficile diagnosis and treatment, a number of commonly used assays for the diagnosis of C. difficile diarrhea were examined. These included an enzyme-linked immunosorbent assay for C. difficile toxin A (ToxA), a CHO cell culture assay for fecal C. difficile (cyto)toxin B, and a lactoferrin latex agglutination assay for fecal lactoferrin (LFLA). We studied 722 fecal specimens submitted by physicians for C. difficile toxin testing at the Salem, Va., Veterans' Affairs Hospital and at the University of Virginia Medical Center in Charlottesville. Charts were reviewed from 123 Veterans' Hospital patients and 114 University of Virginia patients for clinical criteria indicative of C. difficile diarrhea. An increasing titer of CHO cell cytotoxicity was correlated with an increasing likelihood of ToxA positivity (5 to 90%), LFLA positivity (39 to 77%), and clinical agreement (28 to 85%). However, some data indicate that the CHO cell cytotoxicity assay may be nonspecific when positive only at low titers. When the CHO assay result is positive at high titers, it remains the best diagnostic tool. Yet, when it is positive at a low titer, careful interpretation of the results in conjunction with other assays and the clinical setting is warranted, especially in light of new drug-resistant strains of microorganisms.