The phosphohydrolase component of microsomal glucose-6-phosphatase has been identified as a 62 kDa polypeptide by $^{32}P$-labeling of the phosphoryl-enzyme intermediate formed during steady-state hydrolysis. A 62 kDa polypeptide was labeled when disrupted rat hepatic microsomes were incubated with a $^{32}P$-labeled substrate for the enzyme. The reaction was stopped with 1% sodium dodecyl sulfate. Fasted rat liver had at least a five times stronger band intensity than normal liver. [$^{32}P$] glucose-6-phosphate also labeled a similar-sized polypeptide in human and rabbit hepatic microsomes. The $^{32}P$-labeled 62 kDa polypeptide was extracted using SDS-PAGE and reconstituted with membrane lipids. It exhibited glucose-6-phosphatase activity.