Using a specific 13C NMR localization method, 13C label incorporation into the glycogen C1 resonance was measured while infusing [1- 13C]glucose in intact rats. The maximal concentration of [1-13C]glycogen was 5.1 ± 0.6 μmol g-1 (mean ± SE, n = 8). During the first 60 rain of acute hyperglycemia, the rate of 13C label incorporation (synthase flux) was 2.3 ± 0.7 μmol g-1 h-1 (mean ± SE, n = 9 rats), which was higher (p < 0.01) than the rate of 0.49 ± 0.14 μmol g-1 h-1 measured ≥2 h later. To assess whether the incorporation of 13C label was due to turnover or net synthesis, the infusion was continued in seven rats with unlabeled glucose. The rate of 13C label decline (phosphorylase flux) was lower (0.33 = 0.10 μmol g-1 h-1) than the initial rate of label incorporation (p < 0.01) and appeared to be independent of the duration of the preceding infusion of [1-13C]glucose (p > 0.05 for correlation). The results implied that net glycogen synthesis of ~3 μmol g-1 had occurred, similar to previous reports. When infusing unlabeled glucose before [1-13C]glucose in three studies, the rate of glycogen C1 accumulation was 0.46 ± 0.08 μmol g-1 h-1. The results suggest that steady-state glycogen turnover rates during hyperglycemia are ~1% of glucose consumption.