N2,O2'-dibutyryl cyclic guanosine 3':5'-monophosphate has been reported to inhibit the activity of cholecystokinin on several different end organ responses and in several species. Although the mechanism of this inhibition is unclear, competitive antagonism at the level of the receptor has been suggested. We have investigated an alternate possibility, that N2,O2'-dibutyryl cyclic guanosine 3':5'-monophosphate inhibits cholecystokinin action by interacting directly with the peptide while both are in solution. We used antisera with specificities for different regions of cholecystokinin-gastrin peptides and radioiodinated cholecystokinin-33, cholecystokinin-8, and gastrin-17. Our results support the possibility of a soluble interaction between N2,O2'-dibutyryl cyclic guanosine 3':5'-monophosphate and the peptides of cholecystokinin-gastrin family that is specific for the COOH-terminal (receptor binding) region of these peptides, that is dependent on the concentration of N2,O2'-dibutyryl cyclic guanosine 3':5'-monophosphate, and that correlates with concentrations that inhibit biologic activity. The proposed N2,O2'-dibutyryl cyclic guanosine 3':5'-monophosphate-cholecystokinin complex is dispersed by gel filtration chromatography, and is prevented from being formed by certain detergents.