Previously, we reported that the catalytic subunit of protein phosphatase 2A [PP2Ac] undergoes carboxylmethylation [CML] at its C-terminal leucine, and that inhibitors of such a post-translational modification markedly attenuate nutrientinduced insulin secretion from isolated cells. More recent studies have suggested direct inhibitory effects of glucose metabolites on PP2A activity in isolated cells, implying that inhibition of PP2A leads to stimulation of insulin secretion. Since the CML of PP2Ac has been shown to facilitate the holoenzyme assembly and subsequent functional activation of PP2A, we investigated putative regulation by glucose of the CML of PP2Ac in INS-1 cells. Our data indicated a marked inhibition by specific intermediates of glucose metabolism [e.g., citrate and phosphoenolpyruvate] of the CML of PP2Ac in INS-1 cell lysates. Such inhibitory effects were also demonstrable in intact cells by glucose. Mannoheptulose, an inhibitor of glucose metabolism, completely prevented inhibitory effects of glucose on the CML of PP2Ac. Moreover, glucose-mediated inhibition of the CML of PP2Ac was resistant to diazoxide, suggesting that glucose metabolism and the generation of glucose metabolites might control inhibition of the CML of PP2Ac. A membrane-depolarizing concentration of KCl also induced inhibition of the CML of PP2Ac in intact INS cells. Based on these data, we propose that glucose metabolism, and increase in intracellular calcium facilitate inhibition of the CML of PP2Ac, resulting in functional inactivation of PP2A. This, in turn, might retain the key signaling proteins of the insulin exocytotic cascade in their phosphorylated state leading to stimulated insulin secretion.