The continuous cell line of African green monkey kidney, Vero, showed characteristic morphological changes in response to culture filtrates from toxigenic strains ofEscherichia coli. The response compared favorably with that of Y-1 (mouse adrenal) and CHO (Chinese hamster ovary) cells. Vero cells were the simplest and most economical to maintain in the laboratory. Escherichia coli heat-labile enterotoxin (LT) acts on certain cell cultures to induce measurable biochemical changes, e.g., increased adenylate cyclase activity (6, 10, 13, 22), increased intracellular levels of cyclic adenosine 3',5'monophosphate (9, 22), and induced synthesis of A4,3-keto-steroids (1, 2, 4). With Y-1 (mouse adrenal) and CHO (Chinese hamster ovary) cells, these changes are accompanied by alterations in cell morphology and thus provide a simple diagnostic tool (3, 9, 11, 15, 18, 19). The continuous cell line Vero (African green monkey kidney) is also morphologically affected by LT and shows advantages over other assay methods. The study reported here compares the responses of Y-1 (2), CHO (9), and Vero cells to the culture filtrates of various strains ofE. coli. MATERIALS AND METHODS Enterotoxin production. E. coli enterotoxigenic human or porcine strains H10407, B2C, and B7A were obtained from H. L. DuPont (U.S.A.), and P155, P307, 339, and 711 (P307) were from C. Gyles (Canada). Nontoxigenic strains 711 and K-12 were obtained from C. Gyles and V. N. Iyer (Canada), respectively. Erlenmeyer flasks, 150 ml, containing 20 ml of Trypticase soy broth (TSB) or Evans medium (8) were inoculated and mechanically shaken at 37°C. After 16 to 20 h, the cultures were centrifuged at 17,000 x g for 30 min. The supernatants were filtered through 0.45-,um membrane filters (Millipore Corp.) and stored at 4°C until assay that same day. Filtrate dilutions were made in phosphate-buffered saline (PBS), pH 7.0. Cell culture assay. Stocks of the continuous cell lines Y-1, CHO, and Vero, purchased from the American Type Culture Collection, Rockville, Md., were passaged by trypsinization and grown as monolayers at 36°C in a 5% CO2 atmosphere. Y-1 cells were grown in Ham nutrient mixture F10 (Connaught Laboratories Ltd.), CHO cells were grown in Ham nutrient mixture F12 (Grand Island Biological Co.), and Vero cells were grown in medium 199 with