Single molecules of alkaline phosphatase were assayed using capillary electrophoresis laser-induced fluorescence detection. Multiple incubations of individual molecules were performed. Varying the temperature in multiple incubation assay allowed for the determination of the activation energy of catalysis at the single molecule level. Molecules are heterogeneous with respect to both activity and activation energy of catalysis. Partial thermal denaturation of alkaline phosphatase results from the total denaturation of a fraction of the molecules with surviving molecules unaffected rather than a partial decrease in the activity of all the molecules.