Glucose-6-phosphatase is an important enzyme involved in the regulation of concentration of glucose in blood. Steady-state labeling, a method to incorporate $^{32}P$ to the active site of Glucose-6-Phosphatase, was employed to identify the target enzyme. By the steady state labeling about 62-kDa protein was labeled in the microsome from rat liver. The 62-kDa band disappeared in the presence of known inhibitors and competitors of glucose-6-phosphatase. 35-kDa protein was digested protein fragment from the 62-kDa protein. These results showed that the 62-kDa protein was the phosphohydrolase component of glucose-6-phosphatase system.