Abstract 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 (H 2O 2). 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.