Two cell culture systems were used in a study of the biological properties of several bacterial enterotoxins in vitro. By means of one model, in which HeLa cell monolayers were used, cytotoxic effects, in terms of detachment of cells from a glass surface due to cell death, were assayed. By means of the second model, activation of the adenyl cyclase-cyclic adenosine 3′,5′-monophosphate (AMP) system, in terms of increased steroidogenesis by Y-1 adrenal cells (an effect which we have termed cytotonic), was assayed. None of the four toxins manifested both properties. Rather there was a clear segregation into two cytotoxic enterotoxins (Shigella dysenteriae type 1 and Clostridium perjringens) and two cytotonic products (Vibrio cholerae and Escherichia coli). These data raise the possibility that some enterotoxins may not interact with the adenyl cyclase-cyclic AMP system; this possibility has also been suggested by studies of the toxin of S. dysenteriae type 1 in the rabbit small bowel. These cell culture systems may therefore serve as convenient models for the study of the mechanism of action of both classes of enterotoxin.