We have investigated the chemiluminescence signal of the ferrous iron in the presence of the luminol and lucigenin. Ferrous, but not ferric, iron produced a transient signal in the presence of luminol, but not lucigenin. Ferrous iron-induced luminol chemiluminescence was significantly inhibited in a concentration-dependent manner by superoxide dismutase (SOD) and catalase. Specific hydroxyl radical scavengers, mannitol and dimethyl sulfoxide (DMSO), also markedly attenuated the ferrous iron-induced chemiluminescence. Additionally, antioxidants, urate, ascorbate, and methionine produced concentration-dependent significant inhibitions in this chemiluminescence. These results show that the hydroxyl radical generation is dependent on simultaneous formation of superoxide and hydrogen peroxide (H2O2). Ferrous iron does not generate a chemiluminescence signal in the presence of lucigenin suggesting that the formation of a hydroxyl radical is responsible for the luminol chemiluminescence. Thus, the present study has established a simple and inexpensive cell-free screening method for monitoring the scavenging effects of drugs on the hydroxyl radical. (C) 1998 Elsevier Science Inc.