Opposing adenine nucleotide-dependent pathways regulating guanylyl cyclase C (GC-C) in rat intestinal membranes have been identified and characterized. ATP analogues substituted in the 2-position were potent inhibitors of basal and Escherichia coli heat-stable enterotoxin (ST)-stimulated GC-C, independent of the metal cation cofactor present. Inhibition of GC-C was associated with large changes in Vmax but only small changes in the S0.5, suggesting a noncompetitive mechanism. Also, inhibition of GC-C was associated with a concentration-dependent shift from positive to negative cooperativity when manganese served as the cation cofactor. These data support the existence of a noncompetitive allo steric regulatory mechanism mediating adenine nucleotide-dependent inhibition of GC-C. Adenine nucleotides not substituted in the 2-position potentiated the activation of GC-C by ST in intestinal membranes. The potentiating and inhibitory pathways regulating GC-C enzyme activity were separate and distinct. A specific inhibitor (2-chloroadenosine 5'-triphosphate (2ClATP)), was without effect on the potency of a selective activator (adenosine 5'-O-thiomonophosphate (AMPS)) of GC-C. Similarly, AMPS was without effect on the potency of 2ClATP to inhibit GC-C. These data suggest that adenine nucleotide-dependent activation and inhibition are mediated by independent sites which may modulate the second messenger response of GC-C to ST.