Kinetic studies suggested the presence of several forms of NAD-dependent aldehyde dehydrogenase (ALDH) in rat brain. A subcellular distribution study showed that low- and high-Km activities with acetaldehyde as well as the substrate-specific enzyme succinate semialdehyde dehydrogenase were located mainly in the mitochondrial compartment. The low-Km activity was also present in the cytosol (less than 20%). The low-Km activity in the homogenate was only 10-15% of the total activity with acetaldehyde as the substrate. Two Km values were obtained with both acetaldehyde (0.2 and 2000 microM) and 3,4-dihydroxyphenylacetaldehyde (DOPAL) (0.3 and 31 microM), and one Km value with succinate semialdehyde (5 microM). The main part of the aldehyde dehydrogenase activities with acetaldehyde, DOPAL, and succinate semialdehyde, but only little activity of the marker enzyme for the outer membrane (monoamine oxidase, MAO), was released from a purified mitochondrial fraction subjected to sonication. Only small amounts of the ALDH activities were released from mitochondria subjected to swelling in a hypotonic buffer, whereas the main part of the marker enzyme for the intermembrane space (adenylate kinase) was released. These results indicate that the ALDH activities with acetaldehyde, DOPAL and succinate semialdehyde are located in the matrix compartment. The low-Km activity with acetaldehyde and DOPAL, but not the high-Km activities and succinate semialdehyde dehydrogenase, was markedly stimulated by Mg2+ and Ca2+ in phosphate buffer. The low- and high-Km activities with acetaldehyde showed different pH optima in pyrophosphate buffer.