The polymerase chain reaction was used for the detection of Clostridium difficile, the etiologic agent of antibiotic-associated colitis. An upstream primer identical to a coding region (segment I) of the C. difficile 16SrRNA gene and a downstream primer complementary to a highly conserved region of eubacterial 16S rRNA served to amplify a targeted 270-base-pair fragment of genomic DNA. This technique allowed the detection of as fewas 10 C. difficile organisms among 106Escherichia coli bacteria. This level of sensitivity represents a lOO-fold increase over that of conventional anaerobic culture. C. difficile wasdetected in DNAextracted directly from the stools of 23 patients with antibiotic-associated colitis and from those of four patients with diarrhea whosestoolshad beennegativefor C. difficile whenassessedin a cytotoxicity assay. No amplification products were found in the stools of asymptomatic patients. When detected in stools of symptomatic patients, amplification products of C. difficile were confirmed bySouthern blotting with a nonradioactive, horseradish peroxidase-catalyzed, chemiluminescent probing systemin which biotin-labeled oligonucleotides wereused. This system discriminates between C. difficile and similar organisms, such as Clostridium sordellii and Clostridium bifermentans. The combination of the polymerase chain reaction with enzyme-linked probing results in a faster and more sensitive assay for C. diJficile than standard culture.