To understand the anti-atherosclerotic mechanism of the steroid dehydroepiandrosterone (DHEA), the effect of DHEA on rat peritoneal macrophage superoxide generation was studied. Dehydroepiandrosterone (12.5 to 50 μM) inhibited digitonin-stimulated superoxide production in a dose-dependent manner, with 100% inhibition achieved at 50 μM. Dehydroepiandrosterone also inhibited macrophage superoxide production stimulated with phorbol myristate acetate, A23187 (calcium ionophore), sodium fluoride, and arachidonate. Dehydroepiandrosterone did not affect the activity of nicotinamide-adenine dinucleotide phosphate oxidase, which generates superoxide. Dehydroepiandrosterone inhibited superoxide formation in the presence of nicotinamide-adenine dinucleotide phosphate, potassium cyanide, and 2,4-dinitrophinal, suggesting that DHEA does not exert its effects by inhibiting glucose-6-phosphate dehydrogenase activity or mitochondrial respiration. Of the several steroids tested, epiandrosterone was as effective as DHEA in inhibiting macrophage superoxide production. Estrogen, androstenedione, and dihydroxytestosterone showed 25% inhibition, whereas pregnenolone, progesterone, testosterone, etiocholanolone, androstenediol, and DHEA-sulfate had minimal effect. The steroids cortisol and corticosterone had slight stimulatory effect.These results suggest that the anti-atherosclerotic effect of DHEA may be the result of inhibition of superoxide generation in macrophages.