Ammonia assimilation by the plastidic glutamine synthetase/glutamate synthase system requires 2-oxoglutarate (2-OG) as a carbon precursor. Plastids depend on 2-OG import from the cytosol. A plastidic dicarboxylate translocator 1-[2-OG/malate translocator (DiT1)] has been identified and its substrate specificity and kinetic constants have been analyzed in vitro. However, the role of DiT1 in intact plants and its significance for ammonia assimilation remained uncertain. Here, to study the role of DiT1 in intact plants, its expression was antisense-repressed in transgenic tobacco plants. This resulted in a reduced transport capacity for 2-OG across the plastid envelope membrane. In consequence, allocation of carbon precursors to amino acid synthesis was impaired, organic acids accumulated and protein content, photosynthetic capacity and sugar pools in leaves were strongly decreased. The phenotype was consistent with a role of DIT1 in both, primary ammonia assimilation and the re-assimilation of ammonia resulting from the photorespiratory carbon cycle. Unexpectedly, the in situ rate of nitrate reduction was extremely low in alpha-DiT1 leaves, although nitrate reductase (NR) expression and activity remained high. We hypothesize that this discrepancy between extractable NR activity and in situ nitrate reduction is due to substrate limitation of NR. These findings and the severe phenotype of the antisense plants point to a crucial role of DiT1 at the interface between carbon and nitrogen metabolism.