The purpose of this study was to investigate the generation of superoxide anion radical, O−2, by Kupffer cells in vivo in rat liver. Phorbol myristate acetate (PMA) was infused into perfused rat liver which had been continuously infused with 2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo[1,2-a] pyrazin-3-one (MCLA, a highly sensitive luminescence reagent for O2−) and the MCLA luminescence from the liver surface was detected with a sensitive photon counter. Under identical conditions, but without MCLA infusion, PMA and nitro blue tetrazolium were infused to verify generation of O2−; by formation of formazan deposition. Based on MCLA luminescence and fonnazan deposition, both dependent on the O2−; reaction, we have concluded that liver Kupffer cells generate O2−; in vivo in response to stimuli such as PMA. Further, the generation of O2−; by Kupffer cells may be by the same mechanism as displayed by macrophages.