Rat brain mitochondrial aldehyde dehydrogenast isozymes were purified and characterized. There were at last three mitochondrial ALDH isozymes. The soluble ALDH-I was NAD(P)^+ dependent and it has a very low K_m (2.9×10^(-6)M) for succinic semialdehyde but it has a relatively high K_m (10^(-3)M level) for acetaldehyde. However, another soluble ALDH isozyme did not react with succinic semialdehyde. The membrane bound ALDH also has a very low K_m for succinic semialdehyde, however, its reaction rate was slow and its V_(max)/K_m value was only 1/25 that of the soluble ALDH-I. The soluble ALDH-I was significantly inhibited by acetaldehyde. It seemed that ALDH-I might be involved in GABA metabolism related with physiological disturbance caused by over consumption of ethanol.