Reaction microcalorimetry and equilibrium dialysis have been used to study the binding of AMP and IMP to glycogen phosphorylase b (EC 2.4.1.1) at 25 degrees C and pH 6.9. The combination of both techniques has enabled us to obtain some of the thermodynamic parameters for these binding processes. Four binding sites were found to be present in the dimeric active enzyme for both AMP and IMP. The binding to two high-affinity sites, which, in our opinion, correspond to the activator sites, seems to be cooperative. The two low-affinity sites, which would then correspond to the inhibitor sites, appear to be independent when the nucleotides bind to the enzyme. The negative delta G0 of binding/site at 25 degrees C is the result in all cases of a balance between negative enthalpy and entropy changes. The large differences in delta H and delta S0 for the binding of AMP to the activator sites (-27 and -70 kJ mol-1; -22 and -150 J X K-1 mol-1) suggest the existence of rather extensive conformational changes taking place in phosphorylase b on binding with the allosteric activator. Whereas the affinity of AMP for the activator sites is about 1 order of magnitude higher than that of IMP, the affinity of both nucleotides, including their delta H and delta S0 values, seems to be the same for the inhibitor sites.