OBJECTIVE The impact of storage on stability and detection of Clostridium difficile toxins in feces is poorly understood. The objective of this study was to investigate the immunological stability of C. difficile toxins in clinical stool specimens under different storage conditions by evaluating this stability using toxin detection by enzyme immunoassay (EIA). METHODS Stool specimens positive for C. difficile infection (CDI) by quantitative polymerase chain reaction (qPCR) were used for EIA testing with the C. difficile Tox A/B II kit. The EIA-positive specimens were stored aerobically under refrigerated (4-10°C) and frozen (-30°C and -80°C) conditions. Measurement of toxin quantity was conducting using optical density (OD) on days 0, 14, 30, 60, 90, and 120 of storage. RESULTS Clostridium difficile toxins demonstrated good detection in undiluted stool specimens by EIA up to 120 days of storage. Good detection of the toxins was observed in diluted samples at refrigerated and -80°C temperatures. Dilution detrimentally affected toxin detection at -30°C. CONCLUSION Storage of undiluted clinical stool specimens at refrigerated, -30°C, and -80°C temperatures for up to 120 days has no discernible effect on the immunological stability of C. difficile cytotoxins. However, storage at -30°C has a detrimental effect on C. difficile toxin stability in diluted specimens. Infect Control Hosp Epidemiol 2018;39:434-438.