NADPH is an essential cofactor for many enzymatic reactions including glutathione metabolism and fat and cholesterol biosynthesis. We have reported recently an important role for mitochondrial NADP ؉ -dependent isocitrate dehydrogenase in cellular defense against oxidative damage by providing NADPH needed for the regeneration of reduced glutathione. However, the role of cytosolic NADP ؉ -dependent isocitrate dehydrogenase (IDPc) is still unclear. We report here for the first time that IDPc plays a critical role in fat and cholesterol biosynthesis. During differentiation of 3T3-L1 adipocytes, both IDPc enzyme activity and its protein content were increased in parallel in a time-dependent manner. Increased expression of IDPc by stable transfection of IDPc cDNA positively correlated with adipogenesis of 3T3-L1 cells, whereas decreased IDPc expression by an antisense IDPc vector retarded adipogenesis. Furthermore, transgenic mice with overexpressed IDPc exhibited fatty liver, hyperlipidemia, and obesity. In the epididymal fat pads of the transgenic mice, the expressions of adipocyte-specific genes including peroxisome proliferator-activated receptor ␥ were markedly elevated. The hepatic and epididymal fat pad contents of acetylCoA and malonyl-CoA in the transgenic mice were significantly lower, whereas the total triglyceride and cholesterol contents were markedly higher in the liver and serum of transgenic mice compared with those measured in wild type mice, suggesting that the consumption rate of those lipogenic precursors needed for fat biosynthesis must be increased by elevated IDPc activity. Taken together, our findings strongly indicate that IDPc would be a major NADPH producer required for fat and cholesterol synthesis.Abnormal lipid metabolism is frequently associated with obesity and hyperlipidemia. In fat and cholesterol biosynthesis, NADPH is an essential cofactor for numerous enzymes. For instance, 3-L-hydroxylacyl-coenzyme A dehydrogenase and enoyl-coenzyme A reductase in fatty acid synthesis and hydroxymethylglutaryl-coenzyme A reductase, the rate-limiting enzyme in cholesterol biosynthesis, require NADPH for their enzyme activities. It has been demonstrated that glucose-6-phosphate dehydrogenase (G6PDH), 1 6-phosphogluconate dehydrogenase, and malic enzyme are considered as the major enzymes producing cytosolic NADPH (1). Nevertheless, the activities of these enzymes were markedly lower than that of cytosolic NADP ϩ -dependent isocitrate dehydrogenase (IDPc) in the rat liver (1, 2). Consistent with this observation, McLean and co-workers (3) reported that certain adaptive changes in the pentose phosphate pathway dehydrogenases did not take place in parallel with fat synthesis in adipose tissue and suggested that a major source of NADPH for fat synthesis could be IDPc. It is worthy of note that IDPc is expressed mainly in lipogenic tissues such as liver and adipocytes, whereas G6PDH and 6-phosphogluconate dehydrogenase are expressed ubiquitously (4, 5). These data indicate that NADPH-producing IDPc may ...