Various techniques have been employed previously to show that acetaldehyde is primarily oxidized in the mitochondrial matrix of rat liver. In this study, a new approach was tested. Mitochondrial low-Km aldehyde dehydrogenase (ALDH) was partially inactivated and the effect on acetaldehyde oxidation measured. Cyanamide was chosen as the ALDH inhibitor. An enzymatic activation of cyanamide, probably by catalase, was necessary for the drug to inhibit ALDH activity. The level of remaining ALDH activity after cyanamide treatment was correlated with the ability of either rat liver mitochondria or liver slices to oxidize acetaldehyde. Any inhibition of ALDH resulted in a decreased rate of acetaldehyde oxidation, indicating that there is no excess of ALDH in the cell above what is needed to oxidize acetaldehyde. Approximately 15% of the acetaldehyde disappearance at 200 microM was catalyzed by high-Km ALDH, and nearly 30% of the acetaldehyde was lost through binding to cytosolic proteins.