A chromatographic method has been developed which permits the isolation of phosphorylated and dephosphorylated enzymatically active forms of phosphoglucomutase. The enzyme, obtained from rabbit muscle, is chromatographed at pH 7 on carboxymethyl-cellulose, eluting with a linear gradient of sodium phosphate buffer. Inactive protein, if present, passes through the column and is separated from the active enzyme forms which are subsequently eluted.Chromatographed enzyme can be stored at approximately 0°as a slurry in pH 5-buffered ammonium sulfate; or, alternatively, in liquid nitrogen as frozen pellets of neutral buffered solutions. That a homogeneous enzyme of high purity can be obtained by such chromatography was evidenced by the constant specific enzymatic activity exhibited by the fractions belonging to a single peak, by the enzyme's subsequent elution as a single peak upon repeated chromatography, and by its homogeneity in equilibrium ultracentrifugation. The phosphorylated and dephosphorylated nature of the separated phosphoglucomutase components was identified by 32P-substrate labeling experiments. Amino acid analyses were performed, and the composition of the chromatographed phosphoenzyme was compared with that published for phosphoglucomutase in earlier reports.Recent studies concerning the relationship of structural characteristics of phosphoglucomutase to its activity (Koshland etal., 1962), in some cases involving rather small but measurable changes, emphasized the desirability of finding a reproducible method for obtaining enzyme samples of the highest purity. Moreover, according to the mechanism of action proposed by Najjar and Pullman (1954), this muscle enzyme must exist in two catalytically active forms, one a phosphoprotein and the other a nonphosphorylated species. A separation of these two forms would be