The impact of Clostridium difficile fecal loads on diagnostic test results is poorly understood, but it may have clinical importance. In this study, we investigated the relationship between C. difficile fecal load and the results of four assays: a glutamate dehydrogenase (GDH) enzyme immunoassay (EIA), a toxin A/B antigen EIA (ToxAB), a cell culture cytotoxicity assay (CCA), and PCR targeting the tcdB gene. We also compared the PCR cycle threshold (C T ) with the results of quantitative culture using Spearman's rank correlation coefficient. .35 to 7.83), respectively. Group 1 samples had lower fecal loads than those from each of the other groups (P < 0.001). Group 2 samples had lower fecal loads than those from groups 3 and 4 (P < 0.001). There was a significant correlation between PCR C T and fecal loads ( ؍ ؊0.697; P < 0.001). NAP1 strains were associated with the detection of toxins by EIA or CCA (P ؍ 0.041). This study demonstrates an association between C. difficile fecal load and the results of routinely used diagnostic tests.