Experiments were carried out to study the effect of two commonly used glutathione-depleting agents, diethylmaleate and phorone, on the oxidation of acetaldehyde and the activity of aldehyde dehydrogenase. The oxidation of acetaldehyde by intact hepatocytes was inhibited when the cells were incubated with diethylmaleate. Washing and resuspending the cells in diethylmaleate-free medium afforded protection against the inhibition of acetaldehyde oxidation. The oxidation of acetaldehyde by isolated rat liver mitochondria as well as by disrupted mitochondria in the presence of excess NAD+ was inhibited by diethylmaleate or phorone, indicating inhibition of the low-Km aldehyde dehydrogenase. In addition, diethylmaleate inhibited oxidation of acetaldehyde by the high-Km cytosolic aldehyde dehydrogenase. Significant accumulation of acetaldehyde occurred when ethanol was oxidized by hepatocytes in the presence, but not in the absence, of diethylmaleate. Thus, diethylmaleate blocks the oxidation of added or metabolically generated acetaldehyde, analogous to results with other inhibitors of the low-Km aldehyde dehydrogenase such as cyanamide. These results suggest that caution should be used in interpreting the effects of diethylmaleate or phorone on metabolic reactions, especially those involving metabolism of aldehydes such as formaldehyde, because, in addition to depleting glutathione, these agents inhibit the low-Km aldehyde dehydrogenase.