The study investigated the relationship between lipid peroxidation and enzyme inactivation in rat hepatic microsomes and whether prior inactivation of aldehyde dehydrogenase (ALDH) exacerbated inactivation of other enzymes. In microsomes incubated with 2.5 microM iron as ferric sulfate and 50 microM ascorbate, ALDH, glucose-6-phosphatase (G6Pase) and cytochrome P450 (Cyt-P450) levels decreased rapidly and concurrently with increased levels of thiobarbituric acid-reactive substances. Microsomal glutathione S-transferase and nicotinamide adenine dinucleotide phosphate-cytochrome c reductase were little affected during 1 hr of incubation. Addition of reduced glutathione partially protected and N,N'-diphenyl-p-phenylenediamine and butylated hydroxytoluene completely protected microsomes against inactivation of ALDH, G6Pase and Cyt-P450, as well as lipid peroxidation induced by iron and ascorbate. ALDH was more susceptible than G6Pase to inactivation by iron and ascorbate, and was thus an excellent marker for oxidative stress. Inhibition of ALDH by cyanamide injection of rats exacerbated the inactivation of G6Pase in microsomes incubated with 0.1 mM, but not 25 microM 4-hydroxynonenal (4-HN). 4-HN did not stimulate lipid peroxidation. Thus, 4-HN may play a minor role in microsomal enzyme inactivation. In contrast, lipid peroxyl radicals play an important role in microsomal enzyme inactivation, as evidenced by the prevention of both lipid peroxidation and enzyme inactivation by chain-breaking antioxidants.