Using a sensitive Vero (African green monkey kidney) cell model system, studies were performed to further investigate whether Clostridium perfringens enterotoxin acts via disruption of the colloid-osmotic equilibrium of sensitive cells. Enterotoxin was shown to cause a rapid loss of intracellular 86Rb + ( M r approx. 100) with time- and dose-dependent kinetics. The enterotoxin-induced release of intracellular 86Rb + preceded the loss of two larger labels, 51Cr label ( M r approx. 3500) and 3H-labeled nucleotides ( M r less than 1000). The osmotic stabilizers, sucrose and poly(ethylene glycol), differentially inhibited enterotoxin-induced larger label loss versus 86Rb + loss. Further, enterotoxin was shown to cause a rapid influx of 24Na + that was not significantly inhibited by osmotic stabilizers. Additional studies demonstrated that lysosomotropic agents were not protective against characteristic enterotoxin-induced membrane permeability alterations or morphological damage. Taken collectively, these results are consistent with an action for enterotoxin which involves a disruption of the osmotic equilibrium.