When a liver perfusion with nitro blue tetrazolium (NBT) and phorbol myristate acetate (PMA) was performed in carbon tetrachloride (CCl4)-intoxicated rats, formazan deposition was remarkable in macrophages in the necrotic areas of the liver, its intensity varying with the extent of injury. The deposits almost disappeared after addition of Cu(Lys)2, a scavenger of intra- and extracellular superoxide, but were not affected by superoxide dismutase (SOD), which acts extracellularly. The formazan content after incubation with NBT and PMA was higher in macrophages isolated from CCl4-intoxicated liver than in those from normal liver, though their PMA-induced chemiluminescence did not differ. In Corynebacterium parvum-treated liver, both Cu(Lys)2 and SOD reduced the deposits. This method can estimate in situ the ability of hepatic macrophages to produce superoxide and the cellular sites of its production.