An assay for measurement of optimal amounts of glycogen synthase R, the physiologically active form of the enzyme, in liver tissue extracts is described. Tissue extracts enriched in synthase R had a pH profile different from those reported for synthase D and synthase I. In tissue extracts, synthase I had a broad pH optimum but maximal activity was present at pH 7.0-9.0. Synthase D had a sharp pH optimum at pH 8.5 and had little activity at pH 7.0, either in the presence or in the absence of glucose 6-phosphate (G6P). In extracts enriched in synthase R, the pH optimum was 7.0-8.0 without G6P, but 8.0 with G6P. The synthase R activity without G6P rapidly decreased at a higher pH. The proportion of synthase in the physiologically active form traditionally has been reported as an activity ratio based upon the activity in the presence and absence of G6P. The assay has been performed at a single pH. Because of the differences in pH profile, we recommend that the enzyme be measured at pH 7.0 in the absence of G6P and pH 8.5-8.8 in the presence of G6P. In previous assays the substrate UDP-Glc concentration used often has been less than saturating, and the G6P concentration generally has been excessive. A substrate concentration of 11 mM UDP-Glc was found to be necessary for maximal activity. A G6P concentration of 2 mM is adequate for measurement of the D form of the enzyme.