1,2,3-Benzenetricarboxylate (BTA) and n-butylmalonate (BM), specific inhibitors of the mitochondrial tricarboxylate and dicarboxylate carrier, respectively, have been used to study the contribution of citrate export from mitochondria to the accumulation of fat in 3T3-L1 cells. Continuous treatment of the cells with BTA or BM for 5 days after the induction of differentiation caused a significant reduction in fat accumulation in the cells in an inhibitor concentration-dependent manner. These inhibitory effects of BTA and BM were not due to their side effects on DNA replication, since similar inhibition of fat accumulation was not observed with ordinary inhibitors of DNA replication. A similar reduction in fat accumulation was also observed when the cells were treated with BTA or BM for only 2 days just after induction of differentiation. However, interestingly, treatment of the cells with an inhibitor starting 2 days after the induction did not result in reduced fat accumulation. Furthermore, Northern analysis clearly indicated that transcript levels of peroxisome proliferator-activated receptor gamma (PPARgamma) and adipose-type fatty acid binding protein (A-FABP) were well correlated with the levels of fat accumulation. These results clearly indicate the essential role of citrate export from the mitochondrial matrix to the cytosol at the early differentiation stage of 3T3-L1 cells for their effective differentiation into fat cells.