Cytolethal distending toxin (CDT) is a multicomponent bacterial holotoxin that targets most eukarytotic cells causing distension and cell cycle arrest. A number of diverse pathogenic bacterial species associated with diarrhoea, chancroid, chronic hepatitis and periodontal disease produce a CDT. Synthesis of the holotoxin is directed by the expression of three genes, cdtA, cdtB and cdtC. Although the product of the CdtB gene was previously identified as a type I deoxyribonuclease, the functions of the cdtA and cdtC products have not been characterized. Using the periodontal pathogen, Actinobacillus actinomycetemcomitans, we demonstrate that the recombinant product of the CdtA gene binds to the surface of Chinese hamster ovary (CHO) cells. This protein did not induce distension or cytotoxicity when introduced into the cytosol using a lipid-based protein delivery system. Recombinant CdtB and CdtC proteins failed to bind to CHO cells. However, the delivery of either CdtB or CdtC into the cytosol resulted in the characteristic pattern of distension followed by cell death. Based on these results, it appears that the CdtA protein subunit alone is responsible for anchoring the holotoxin to the cell surface. The CdtC subunit, in concert with CdtB, contributes to the cytotoxic activities of the holotoxin. The specific mechanism of CdtC cytotoxicity is currently unknown.