A rapid assay was developed for detection of the Clostridium difficile enterotoxin gene in stool specimens by means of the polymerase chain reaction (PCR). The PCR primers amplified a 63-bp repetitive sequence of the enterotoxin gene, thereby generating a distinctive ladder pattern of DNA bands following electrophoresis. Crude DNA extracts from stools containing C. difficile produced one (63-bp) or more bands of the characteristic ladder. Of 172 stool specimens from 58 patients, 37 gave positive results by culture (15 specimens) or cytotoxin assay (36 specimens). When 36 available "positive" specimens were tested by the PCR assay, 34 (94%) gave positive results--24 by direct testing, and 10 after extraction of DNA by the Qiagen procedure. Insufficient material of the remaining two specimens was available for DNA extraction. Of 21 stools "negative" for C. difficile by culture or cytotoxin assay, one gave a positive result by PCR and seven produced atypical bands. The rapid PCR detection technique for C. difficile was more sensitive than standard culture, and of a sensitivity similar to cytotoxin testing. The method has the potential for adoption in routine laboratory practice.