The in vitro oxidation of benzaldehyde and acetaldehyde was studied in liver samples from disulfiram-treated and control rats. With 25 μM substrate, both cytosol and mitochondria appeared to make a nearly equal contribution to the oxidation of benzaldehyde, whereas ca. 90% of acetaldhyde oxydation occurred in mitochondria. When the K_m values for benzaldehyde with aldehyde dehydrogenase (ALDH) were determined, two K_m values (3 and 120 μM) were obtained with mitochondria, but only a single K_m value (25 μM) was obtained with the cytosolic fraction. The relatively high K_m (2.9 mM) found with microsomes makes it unlikely that microsomes are important in the oxidation of benzaldehyde. In intact mitochondria, with 200 μM acetaldehyde or benzaldehyde the matrix space enzyme accounted for 77 and 62%, respectively, of the total ALDH activity. When the activity was determined in a mixture containing both substrates, the activity was found not to be additive, indicating that both substrates are oxidized by the same matrix space enzyme. With subcellular fractions, from livers of disulfiram-treated and control rats, a greater degree of inhibition of ALDH was obtained when acetaldehyde was a substrate compared to that with benzaldehyde in cytosol and mitochondria. Microsomal ALDH was not inhibited by disulfiram. In liver slices from rats given disulfiram, a statistically significant inhibition was found when either 25 or 250 μM acetaldehyde was used (46 and 33%). With benzaldehyde, a significant inhibition (24%) was observed only with the lower substrate concentration. Finding that both mitochondrial fractions and slices were less inhibited at the higher substrate concentration implies that the high K_m enzyme is not inhibited. It can be concluded that, in rat, disulfiram inhibiting liver ALDH not only affects oxidation of acetaldehyde, but also that of benzaldehyde.