Escherichia coli strains expressing cytolethal distending toxin (CDT) cause elongation of CHO cells at 24 h, followed by progressive cellular distention and death for up to 120 h. Similar distention and cytotoxicity are seen in HeLa, HEp-2, and, to a lesser extent, Vero cells. The initial elongation in CHO cells is indistinguishable from that caused by E. coli heat-labile toxin (LT). In contrast to those from LT strains, supernatants from these strains have no effect on Y-1 adrenal cells. TnphoA was introduced into CDT-positive E. coli E6468/62 (O86:H34), isolated from a child with diarrhea, and 13 CDT-negative transconjugants were identified. DNA probes constructed from DNA flanking the TnphoA insertion sites of CDT-negative mutants were used to identify a CDT-positive clone from an E6468/62 genomic library with a 5.5-kb insert. Exonuclease deletions were created and assayed in CHO cells. In this manner, a 2.3-kb CDT-active region was defined, and the nucleotide sequence was determined. Sequence analysis identified three open reading frames (ORFs), designated cdtA, cdtB, and cdtC. These contain 711, 819, and 570 bp, respectively, and encode polypeptides with predicted molecular masses of 25.5, 29.8, and 20.3 kDa, respectively. Each ORF has a putative signal sequence, and there are 4-bp overlaps between cdtA and cdtB and between cdtB and cdtC. The nucleotide and predicted amino acid sequences have no significant homology with those of any previously reported genes or proteins. By in vitro transcription-translation and an anti-alkaline phosphatase immunoblot, native proteins and/or fusion proteins corresponding to each ORF were identified.