Acetyl-CoA carboxylase (ACC) catalyzes the formation of malonyl-CoA, a precursor in the biosynthesis of longchain fatty acids, which have been implicated in physiological insulin secretion. The catalytic function of ACC is regulated by phosphorylation (inactive)؊de-phosphorylation (active). In this study we investigated whether similar regulatory mechanisms exist for ACC in the pancreatic islet ␤-cell. ACC was quantitated in normal rat islets, human islets, and clonal ␤-cells (HIT-15 or INS-1) using a [ 14 C]bicarbonate fixation assay. In the ␤-cell lysates, ACC was stimulated by magnesium in a concentration-dependent manner. Of all the dicarboxylic acids tested, only glutamate, albeit ineffective by itself, significantly potentiated magnesium-activated ACC in a concentration-dependent manner. ACC stimulation by glutamate and magnesium was maximally demonstrable in the cytosolic fraction; it was markedly reduced by okadaic acid (OKA) in concentrations (<50 nmol/l) that inhibited protein phosphatase 2A (PP2A). Furthermore, pretreatment of the cytosolic fraction with anti-PP2A serum attenuated the glutamate-and magnesium-mediated activation of ACC, thereby suggesting that ACC may be regulated by an OKA-sensitive PP2A-like enzyme. Streptavidin-agarose chromatography studies have indicated that glutamate-and magnesium-mediated effects on ACC are attributable to activation of ACC's dephosphorylation; this suggests that the stimulatory effects of glutamate and magnesium on ACC might involve activation of an OKA-sensitive PP2A-like enzyme that dephosphorylates and activates ACC. In our study, 5-amino-imidazolecarboxamide (AICA) riboside, a stimulator of AMP kinase, significantly inhibited glucosemediated activation of ACC and insulin secretion from isolated ␤-cells. Together, our data provide evidence for a unique regulatory mechanism for the activation of ACC in the pancreatic ␤-cell, leading to the generation of physiological signals that may be relevant for physiological insulin secretion. Diabetes 50:1580 -1587, 2001