We investigated the interactions of insulin and okadaic acid, an inhibitor of protein phosphatases type-2A and type-1, on glycogen synthesis in rat, guinea pig and rabbit hepatocytes. Insulin stimulated glycogen synthesis in rat and guinea pig but not in rabbit hepatocytes. In rat and guinea pig hepatocytes, the stimulation of glycogen synthesis by insulin was inhibited by low concentrations of okadaic acid (2.5–5 nM), which did not inhibit glycogen synthesis in the absence of insulin. In rabbit hepatocytes, insulin increased the sensitivity of glycogen synthesis to inhibition by low concentrations of okadaic acid even though it did not stimulate glycogen synthesis, and in the presence of insulin and okadaic acid (5 nM) glycogen synthesis was significantly lower than in the presence of okadaic acid alone. An increase in extracellular pH from 7.4 to 7.8 in a bicarbonate-free buffer, decreased the concentration of okadaic acid causing half-maximal inhibition of glycogen synthesis. It is suggested that an increase in cytosolic pH may be one mechanism by which insulin alters the sensitivity of glycogen synthesis to phosphatase inhibition.