ABSTRACT Enterotoxigenic Escherichia coli is a major cause of acute diarrheal illness worldwide and is responsible for high infant and child mortality rates in developing nations. Two types of enterotoxins, one heat labile and the other heat stable, are known to cause diarrhea. The expression of soluble heat-labile toxin is subject to catabolite (glucose) activation, and three binding sites for cAMP receptor protein (CRP or CAP) were identified upstream and within the toxin promoter by DNase I footprinting. One CRP operator is centered at −31.5, thus encompassing the promoter's −35 hexamer. Potassium permanganate footprinting revealed that the occupancy of this operator prevents RNA polymerase from forming an open complex in vitro. However, the operator centered at −31.5 is not sufficient for full repression in vivo because the deletion of the other two CRP binding sites partially relieved the CRP-dependent repression of the heat-labile toxin promoter. In contrast to heat-labile toxin, CRP positively regulates the expression of heat-stable toxin. Thus, the conditions for the optimal expression of one enterotoxin limit the expression of the other. Since glucose inhibits the activity of CRP by suppressing the pathogen's synthesis of cyclic AMP (cAMP), the concentration of glucose in the lumen of the small intestine may determine which enterotoxin is maximally expressed. In addition, our results suggest that the host may also modulate enterotoxin expression because cells intoxicated with heat-labile toxin overproduce and release cAMP.