Interactions of the drug carbamazepine with the serum protein alpha(1)-acid glycoprotein (AGP) were examined by high-performance affinity chromatography. Frontal analysis studies with an immobilized AGP column and control column indicated carbamazepine had both low-affinity interactions with the support and high-affinity interactions with AGP. When a correction was made for binding to the support, the association equilibrium constant measured at pH 7.4 and 37 degrees C for carbamazepine with AGP was 1.0 (+/-0.1) x 10(5) M(-1), with values that ranged from 5.1 to 0.58 x 10(5) M(-1) in going from 5 to 45 degrees C. It was found in competition studies that these interactions were occurring at the same site that binds propranolol on AGP. Temperature studies indicated that the change in enthalpy was the main driving force for the binding of carbamazepine to AGP. These results provide a more complete picture of how carbamazepine binds to AGP in serum. This report also illustrates how high-performance affinity chromatography can be used to examine biological interactions and drug-protein binding in situations in which significant interactions for an analyte are present with both the chromatographic support and an immobilized ligand.