The presence of macroamylasemia can be suspected when a patient has low renal clearance of amylase relative to creatinine (Cam/Ccr). However, the Cam/Ccr is seldom reduced to the extent expected given the fraction of amylase that appears to be bound in the macroamylase complex by gel filtration studies at 3°C. Therefore, we tested the possibility that the binding of amylase in macroamylase complexes was temperature dependent and that binding at physiological temperatures might be less than at 3°C. Gel filtration of five macroamylase-containing sera was carried out at temperatures of 3°, 25°, 37°, 43°, and 49°C. Binding of amylase in the complex decreased as the temperature increased with virtually all amylase bound at 3°C and virtually no amylase bound at 49°C. The fraction of the amylase that bound at 37°C was approximately what would have been predicted from the Cam/Ccr of each of the five subjects. We conclude that amylase binding in macroamylase complexes is extremely temperature sensitive and appreciable changes in binding may occur over the physiological temperature range of 37-41°C.