A new heat-labile toxin cytolethal to CHO, Vero, HeLa, and HEp-2 cells and negative in Y-1 cells has been demonstrated in culture filtrates of many strains of Campylobacter spp. This new toxin was termed a cytolethal distending toxin (CLDT) to reflect the progressive cell distention and eventual cytotoxicity observed in all sensitive tissue cells. CLDT was distinct from previously reported cytotoxins and cholera-like enterotoxin produced by some Campylobacter spp. Since CHO elongation induced by either the Campylobacter enterotoxin or CLDT could not be differentiated after 24 h incubation, continuation of the assay for 96 h was essential for observation of CLDT-associated progressive morphological changes and cytolethal events. Specific assay conditions were required for demonstration of CLDT in Vero, HeLa, and HEp-2 cells. A 31-fold increase in cyclic AMP levels was observed in CHO cells exposed for 24 h to CLDT of catalase negative or weak positive Campylobacter. CLDT was detected in culture filtrates from strains of Campylobacter jejuni, C. coli, C. fetus subsp. fetus, C. laridis and catalase negative or weak positive Campylobacter. Of 718 strains investigated from both human and animal isolations, 295 (41%) were found to produce this toxin. Campylobacter CLDT was negative in adult rabbit ligated ileal loops, suckling mouse and rabbit skin tests. Hemorrhagic responses were observed in rat ligated ileal loop tests of CLDT-positive cultures. The new CLDT toxin could only be neutralized by homologous rabbit antitoxin, was trypsin-sensitive, nondialyzable and over 30,000 in molecular weight. CLDT-producing strains were observed in many serogroups and biotypes of Campylobacter spp. The strains tested originated from many countries and no clear association of toxigenicity with serotype or biotype could be established.