To assess whether hepatic glycogen is actively turning over under conditions which promote net glycogen synthesis we perfused livers from 24-h fasted rats with 20 mM D-[1-13C]glucose, 10 mM L-[3-13C]alanine, 10 mM L-[3-13C]lactate, and 1 microM insulin for 90 min followed by a 75-min "chase" period with perfusate of the same composition containing either 13C-enriched or unlabeled substrates. The peak height of the C-1 resonance of the glucosyl subunits in glycogen was monitored, in real time, using 13C NMR techniques. During the initial 90 min the peak height of the C-1 resonance of glycogen increased at almost a constant rate reflecting a near linear increase in net glycogen synthesis, which persisted for a further 75 min if 13C-enriched substrates were present during the "chase" period. However, when the perfusate was switched to the unenriched substrates, the peak height of the C-1 resonance of glycogen declined in a nearly linear manner reflecting active glycogenolysis during a time of net glycogen synthesis. By comparing the slopes of the curve describing the time course of the net [1-13C] glucose incorporation into glycogen with the rate of net loss of 13C label from the C-1 resonance of glycogen during the "chase" period we estimated the relative rate of glycogen breakdown to be 60% of the net glycogen synthetic rate. Whether this same phenomenon occurs to such an appreciable extent in vivo remains to be determined.