An enzyme-linked immunoassay (ELISA) with 11 Clostridium difficile serogroup-specific antisera was applied for serogrouping of C. difficile colonies from 314 consecutive positive fecal samples. Two hundred forty-nine strains (79%) were correctly serogrouped, 57 (18%) belonged to serogroups other than the 11 which were evaluated and gave a negative reaction with all antisera, and 8 isolates (2.5%) did not react with their corresponding antisera. ELISA is a rapid and reliable method for serogrouping C. difficile and should allow for the automation of this procedure.