Enzyme immunoassays for detection of Clostridium difficile toxins A and B were developed with use of a double-sandwich microtiter plate format. Each assay was specific for its respective toxin and was sensitive to 0.1 ng of toxin. Neither assay was reactive with 13 other species of clostridia. One hundred fifty fecal specimens submitted for tissue culture cytotoxicity assay were evaluated by enzyme-linked immunosorbent assay (ELISA). Of the 79 tissue culture-positive specimens, 72 (91%) were positive in the A assay, 63 (80%) were positive in the B assay, and 75 (95%) were positive in either assay. Specimens with tissue culture titers of greater than or equal to 10(3) were uniformly positive in both assays. The specificities of the toxin A and B ELISAs were 98.6% and 100%, respectively. An ELISA for both toxins could serve as a substitute for the tissue culture cytotoxicity assay.