We reviewed the results of repeated sample submissions within a 7-day time frame for Clostridium difficile toxin testing. A total of 2,940 samples were tested during a 3-month period using a cell culture cytotoxicity assay (CCCA). The results from all second samples (n = 1,101) were concordant with the original test result. In only two cases (0.8%; n = 247) was a third sample positive when the first two samples were negative. In this study, submission of multiple samples for CCCA did not increase detection of Clostridium difficile infection.