The rate of p-nitroanisole O-demethylation is markedly inhibited by ethanol. To evaluate a role of acetaldehyde in the inhibition by ethanol, a comparison was made of the effects of ethanol and acetaldehyde on the metabolism of p-nitroanisole by isolated liver cells. No effect on the metabolism of p-nitroanisole was found at low concentrations of acetaldehyde (<0.5 mm), whereas inhibition occurred at high concentrations (1 mm). In fact, acetaldehyde was not any more inhibitory than crotonaldehyde, which is a poor substrate for the low-Km mitochondrial aldehyde dehydrogenase. Cyanamide, an inhibitor of acetaldehyde oxidation, did not prevent the inhibition by ethanol. Crotonol, an alcohol that does not change the mitochondrial redox state, in contrast to ethanol, proved to be a more effective inhibitor of the metabolism of p-nitroanisole than ethanol. Greater sensitivity to crotonol was also found in isolated microsomes and may reflect hydrophobic effects by crotonol, relative to ethanol. These results suggest that although high levels of acetaldehyde can be inhibitory, physiological levels of acetaldehyde did not affect the metabolism of p-nitroanisole. It is unlikely that acetaldehyde itself plays a major role in the mechanism by which ethanol inhibits the metabolism of p-nitroanisole. The inhibition of p-nitroanisole O-demethylation by ethanol was prevented by pyruvate or fructose, but not by xylitol, sorbitol, or lactate. All these substrates by themselves stimulated metabolism of p-nitroanisole. Pyruvate and glyceraldehyde (which arises from the metabolism of fructose) can oxidize cytosolic NADH. These results suggest that the generation of cytosolic NADH from the oxidation of ethanol, the subsequent requirement for substrate shuttles to transfer NADH into the mitochondria, and redox inhibition of the citric acid cycle, interfere with the transport of NADPH out of the mitochondria, and consequently with drug metabolism.