Abstract: Although mitochondria have been the object of intensive study over many decades, some aspects of their metabolism remain to be fully elucidated, including the l-lactate metabolism. We review here the novel insights arisen from investigations on l-lactate metabolism in mammalian, plant and yeast mitochondria. The presence of l-lactate dehydrogenases inside mitochondria, where l-lactate enters in a carrier-mediated fashion, suggests that mitochondria play an important role in l-lactate metabolism. Functional studies have demonstrated the occurrence of several l-lactate carriers. Moreover, immunological investigations have proven the existence of monocarboxylate translocator isoforms in mitochondria.