FAD-dependent malate dehydrogenase, a phospholipid-requiring enzyme, was homogeneously purified from the particulate fraction of Mycobacterium sp. strain Takeo. The isolated enzyme contains no FAD and few phospholipid, and has a specific activity of 300-360 units/mg of protein. In the assay system without addition of phospholipid (cardiolipin), the enzyme activity was only about 3% of maximum activity. The molecular weight was estimated to be 51 000-55 000 by four methods. Titration by p-chloromercuribenzoate revealed the presence of one cysteine residue/mol of enzyme. The isoelectric point was found to be pH 6.9 by isoelectric focusing. From circular dichroism spectral data, the enzyme protein was found to contain alpha-helix structure of 24%.