Diethyl maleate (DEM), a well-known glutathione (GSH) depletor, causes a dose-dependent increase in hepatic ornithine decarboxylase (ODC) activity as well as heme oxygenase activity in rats. Considering the important role ODC has in polyamine biosynthesis in response to endogenous and exogenous stimuli, further extensive studies on the effect of DEM on ODC in relation to its GSH-depleting effect were carried out. Specifically, concomitant with the profound decrease in GSH content, the higher dose of DEM (1284 mg/kg) caused a marked increase in ODC activity (about 1000 times that of the control) at 12 hr after its administration. DEM at this dose also caused a marked increase in heme oxygenase activity, but the effects on cytochrome P-450 content and aminopyrine demethylase activity were less extensive. The increases in ODC and heme oxygenase activities evoked by DEM were almost completely blocked by pretreatment of rats with either actinomycin D or cycloheximide. Parallel to the increase in ODC activity, DEM caused a profound increase in putrescine content in the liver, while the agent reduced spermine content. The administrations of alpha-difluoromethylornithine and 1,3-diaminopropane resulted in the inhibition of DEM-mediated induction of ODC, but not heme oxygenase. In contrast, methylglyoxal bis(guanylhydrazone) inhibited the induction of both ODC and heme oxygenase evoked by DEM. The DEM-induced ODC exhibited two phases of decay with the prolonged half-lives of 26 and 223 min. Additionally, the elution profile from DEAE-Sepharose CL-6B column chromatography of cytoplasmic fraction from DEM-treated rat liver exhibited two peaks of ODC activity. These findings add new insight into the biochemical effect of DEM on hepatic polyamine metabolism in addition to its GSH-depleting effect.