A counterimmunoelectrophoresis (CIE) technique which reacted positively with culture filtrates of Clostridium difficile was developed and compared with a cytotoxicity assay in human embryonic lung cell cultures. CIE, employing C. sordellii antitoxin, detected 17 of 17 C. difficile strains. Of those positive by CIE, 13 were cytotoxic in cell culture. Fourteen Clostridium species other than C. difficile, C. sordellii, and C. bifermentans were negative by CIE. C. sordellii and C. bifermentans gave positive CIE results but were not cytotoxic. Similar sensitivity of toxin detection was observed for both methods. Optimal conditions for performing CIE included use of 48-h chopped meat-glucose broth cultures as the antigen source, use of a 10x-concentrated U.S. Standard C. sordillii antitoxin, and electrophoresis for 1.5 h in 0.05 M tris(hydroxymethyl)aminomethane-barbital-sodium barbital, pH 8.8, at a constant current of 6 mA/slide. CIE appears to be a suitable alternative to the cytotoxicity assay and may serve as a means for presumptive identification of C. difficile.