We present evidence for the presence of the folate metabolism enzyme methenyltetrahydrofolate synthetase (MTHFS) in mitochondria. MTHFS activity was identified in the matrix of mitochondria purified from human liver biopsies. Mitochondrial and cytoplasmic MTHFS specific activities are similar, 85% of the total cellular MTHFS activity is in the cytoplasm and both native enzymes have similar molecular weights (approximately 25 kDa). Studies using purified mitochondrial MTHFS from CA46 human Burkitt lymphoma cells reveal that mitochondrial MTHFS behaves kinetically like the cytoplasmic enzyme with K m values of 4.7, 0.8 and 22 μM respectively for (6R,S)-5-formyltetrahydrofolate monoglutamate, (6S)-5-formyltetrahydrofolate pentaglutamate and ATP. This finding adds to previous observations that various folate-dependent enzymes reside in the mitochondria of eucaryotic cells. Intracellular tetrahydrofolate metabolism is highly compartmentalized and mitochondrial MTHFS activity is necessary for the entry of mitochondrial 5-formyltetrahydrofolate into the mitochondrial folate pool.