Chromatography of crude rat muscle extracts on CM-cellulose resulted in separation of glucose-6-phosphate isomerase (GPI) into two active peaks (designated types I and II in order of elution). Incubation of type I and type II at pH 6.0, 7.0, or 8.0 in the presence and absence of KCI revealed that one type of GPI is converted to the other type under certain conditions and that the two types are interconvertible by changing the incubation conditions. The two types were similar in molecular weight, kinetic property, and optimum pH. A remarkable difference was observed in pH-activity profile: the activity of type I, but not type II, was detectable at pH 6-7. The present results suggest that rat muscle has two types of GPI which are interconvertible and that the two types may be conformational variants of the same enzyme. It is conceivable that the interconversion between the two types is related to the regulation of glycolysis at lower intracellular pHs.