Addition of 60 mM glucose caused a similar partial activation of glycogen synthase in hepatocytes isolated from overnight fasted Wistar rats and from fasted lean Zucker (Fa/fa?) rats. In contrast, the activation went rapidly to completion in cells from fasted obese (fa/fa) rats. Subsequent addition of 4 microM microcystin, a potent inhibitor of type 1 and type 2A protein phosphatases, induced a rapid inactivation of glycogen synthase, which occurred at a similar rate in all three types of hepatocytes. This suggests that the super-activation of glycogen synthase in hepatocytes from fasted obese rats is not due to a lower synthase kinase activity. Glycogen synthase phosphatase was quantitatively assayed in broken-cell preparations from the same livers, with exogenous synthase b as substrate. The synthase phosphatase activity in the fa/fa livers was 3-fold higher than that in the livers from both lean Zucker rats and Wistar rats. This difference has to be attributed to an increased synthase phosphatase activity of the glycogen-bound protein phosphatase-1 in livers of fasted obese rats. The results suggest that in the latter animals the available insulin exceeds the insulin resistance of the liver. The resulting overexpression of the insulin-dependent synthase-phosphatase-1G activity may explain the super-activation of glycogen synthase in response to a glucose challenge.