Inositol 1,4,5-trisphosphate (IP3) receptors (IP3R) are intracellular Ca2+ channels. Their opening is initiated by binding of IP3 to the IP3-binding core (IBC; residues 224-604 of IP(3)R1) and transmitted to the pore via the suppressor domain (SD; residues 1-223). The major conformational changes leading to IP3R activation occur within the N terminus (NT; residues 1-604). We therefore developed a high-throughput fluorescence polarization (FP) assay using a newly synthesized analog of IP3, fluorescein isothiocyanate (FITC)-IP3, to examine the thermodynamics of IP3 and adenophostin A binding to the NT and IBC. Using both single-channel recording and the FP assay, we demonstrate that FITC-IP3 is a high-affinity partial agonist of the IP3R. Conventional [H-3]IP3 and FP assays pro-vide similar estimates of the K D for both IP3 and adenophostin A in cytosol-like medium at 4 degrees C. They further establish that the isolated IBC retains the ability of full-length IP3R to bind adenophostin A with similar to 10-fold greater affinity than (IP3). By examining the reversible effects of temperature on ligand binding, we established that favorable entropy changes (T Delta S) account for the greater affinities of both ligands for the IBC relative to the NT and for the greater affinity of adenophostin A relative to IP3. The two agonists differ more substantially in the relative contribution of Delta H and T Delta S to binding to the IBC relative to the NT. This suggests that different initial binding events drive the IP3R on convergent pathways toward a similar open state.