The binding of 125I-labeled Escherichia coli heat-stable enterotoxin B to rat intestinal epithelial cells was unsaturable and nonspecific, at concentrations well above that required to mediate biological events. Following its interaction with intestinal cells, approximately 50-80% of heat-stable enterotoxin B remained stably associated with the cells, implying that it was partitioned into the membrane and/or internalized by the cell. The toxin bound with different affinities to lipids isolated from intestinal epithelial cells, phospholipids, glycolipids, neutral lipids and to model membrane vesicles containing negatively charged lipids. These results indicate that heat-stable enterotoxin B utilizes the membrane bilayer, rather than a surface protein or glycoprotein in modulating toxin-induced enterotoxicity.