An NAD-linked aldehyde dehydrogenase which in addition to aliphatic and aromatic aldehydes, metabolizes aminoaldehydes and betaine aldehyde, has been purified to homogeneity from male Sprague–Dawley rat liver mitochondria. The properties of the rat mitochondrial enzyme are similar to those of a rat liver cytoplasmic betaine aldehyde dehydrognase and the human cytoplasmic E3 isozyme. The primary structure. of four tryptic peptides were also similar; only one difference in primary structure was observed. The close similarity of properties of the cytoplasmic with the mitochondrial form suggest that the cytoplasmic and mitochondrial betaine aldehyde dehydrogenase may be coded for by the same nuclear gene. Investigation of the mitochondrial form by isoelectric focusing resulted in visualization of multiple forms, different from those seen in the cytoplasm suggesting that the enzyme may be processed in the mitochondria.