The acid-fast lipid of Mycobacterium tuberculosis, which is considered to be important for the manifestation of its virulence, has very intimate relation with the concentration of glycerol in culture media. In addition to this, glycerol plays a very important role in the metabolism of carbon sources of Mycobacterium tuberculosis. In consideration of these facts, the author studied the dehydrogenase activities of the cell-free extract and of its outer and inner solutions of the dialyzate of Mycobacterium tuberculosis cultured in each of Sauton's liquid media containing glycerol and glucose in place of glycerol. The results are briefly summarized as follows: I) The dehydrogenase of the cell-free extract of H37Rv and H37Ra, particularly of H37Rv, cultured in Sauton's glycerol media are stronger than those cultured in glucose media. 2) For the dehydrogenation of lactate and succinate by the cell-free extract, the addition of the outer solution of dialysis is not needed. In cases of malate and histidine, however, the outer solution is highly needed. 3) The lactic dehydrogenase of the cell-free extract obtained by disintegration in the mortar and centrifugation is very unstable to heating.