The dicarboxylate carrier (DIC) is an integral membrane protein that catalyses a dicarboxylate–phosphate exchange across the inner mitochondrial membrane. We generated a yeast mutant lacking the gene for the DIC. The deletion mutant failed to grow on acetate or ethanol as sole carbon source but was viable on glucose, galactose, pyruvate, lactate and glycerol. The growth on ethanol or acetate was largely restored by the addition of low concentrations of aspartate, glutamate, fumarate, citrate, oxoglutarate, oxaloacetate and glucose, but not of succinate, leucine and lysine. The expression of the DIC gene in wild-type yeast was repressed in media containing ethanol or acetate with or without glycerol. These results indicate that the primary function of DIC is to transport cytoplasmic dicarboxylates into the mitochondrial matrix rather than to direct carbon flux to gluconeogenesis by exporting malate from the mitochondria. The ΔDIC mutant may serve as a convenient host for overexpression of DIC and for the demonstration of its correct targeting and assembly.