The binding of 16 derivatives of cAMP to the surface of Dictyostelium discoideum cells was analyzed. The binding affinity is strongly reduced (more than 14.5 kJ/mol) if a cAMP derivative is no longer able to form a hydrogen bond at N6H2, or at O3’. Decreasing polarity of the base moiety is closely correlated to increasing binding affinity to the cAMP receptor (r = 0.98, p < 0.1%). Based on these results we propose that cAMP is bound to the receptor via hydrogen bonds at N6H2 and O3', and that the adenine moiety is bound in a hydrophobic cleft of the receptor. A stereospecific interaction between the receptor and the phosphate moiety of cAMP has not been observed. The first detectable response of D. discoideum cells to cAMP is an increase of intracellular cGMP levels. A close correlation exists between the binding affinity of the cAMP derivatives and the potency to induce a cGMP response (r = 0.97, p < 0.01%). There are two exceptions; both derivatives are modified in the exocyclic oxygen atoms of the phosphate moiety (sulfur in equatorial position or dimethylamino in apical position). These derivatives bind with approximately the same affinity as their stereoisomers (respectively sulfur in apical or dimethylamino in equatorial position), but in contrast to their stereoisomers, they do not induce elevations of cGMP levels. This suggests that, in addition to the binding interactions mentioned above, activation of the receptor requires a stereospecific interaction between the receptor and the phosphate moiety of cAMP. Quantum-chemical calculations suggest that this activating interaction is a covalent bond between cAMP and the receptor.