A PCR assay that uses primers whose sequences were obtained from the published sequence of the cdt-III gene was developed to determine the frequencies of the cdt-I, cdt-II, and cdt-III genes in Escherichia coli isolates from humans and animals. E. coli isolates producing cytolethal distending toxin (CDT) were infrequently detected. The cdt-I gene was preferentially detected in strains with the cnf1 gene, while the cdt-III gene was found in strains carrying the cnf2 gene. The cdt-III genotype was more prevalent in animal isolates, while the cdt-I and cdt-II genotypes were more evident in human isolates. The presence of further cdt gene variants was indicated by the presence of toxin activity in cell culture in the absence of PCR amplification of the cdt-I, cdt-II, or cdt-III gene.