In the genome of the marine diatom-Thalassiosira pseudonana, there are several putative genes encoding enzymes potentially constitute a classical C4 type biochemical CO2-concentrating mechanism. Two genes encode a carboxylation enzyme phosphoenolpyruvate carboxylase (PEPC)1 and PEPC2; and another two encode decarboxylation enzymes, NAD(+)-dependent malic enzyme (NAD-ME) and phosphoenolpyruvate carboxykinase (PEPCK). These genes were tagged by the enhanced-green fluorescence protein, egfp, ligated in the transformation vector, and transformed into the cells of T. pseudonana for localization of GFP fusion products. The PEPC1:GFP fusion was localized at the matrix of the periplastidal compartment, while the PEPC2:GFP fusion was localized at the mitochondria. The NAD-ME:GFP fusion was localized in the cytosol and the PEPCK:GFP fusion at the mitochondria. The transcripts level of NAD-ME was extremely low, and PEPCK transcript was significantly induced under the dark, suggesting that PEPCK is involved in the dark metabolism such as respiration and amino acid metabolism in the mitochondria. Treatments of low-CO2grown T. pseudonana cells with inhibitors for PEPCK and PEPC efficiently dissipated the maximum rate of photosynthesis while these treatments did not affect high-affinity photosynthesis. These data strongly suggest that classical C4 enzymes play little role in the CCM in T. pseudonana.