Enterotoxigenic Escherichia coli elaborate a heat-stable enterotoxin that causes diarrhea in humans and animals. The primary event in the diarrheal cascade is the binding of this enterotoxin to specific receptors on enterocytes and activation of guanylyl cyclase. Two intestinal cell lines, Caco-2 and IEC-6, were tested for the presence of these receptors. Although both cell lines exhibited specific binding, only the Caco-2 cell line responded to heat-stable enterotoxin with increased guanylyl cyclase activity. Cloning and expression studies confirmed that the receptor present in Caco-2 cells is a homologue of guanylyl cyclase C, a known transmembrane heat-stable enterotoxin receptor. Expression of the receptor in differentiating Caco-2 cells increases with cell maturation, indicating that these cells are a suitable model for future studies. However, Northern and polymerase chain reaction analyses demonstrated that guanylyl cyclase C is not expressed in IEC-6 cells, strongly suggesting the presence of a novel heat-stable enterotoxin receptor that is not coupled to guanylyl cyclase activity.