Cytotoxic actions of alloxan are thought to be caused by the hydroxyl radical (HO·) generated in a cyclic reaction involving alloxan and its reduction product, alloxan radical (HA·). The generation of HA· and the oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) were observed in the reaction system of alloxan with NADPH in the presence of purified NADPH : cytochrome P-450 reductase [EC 1.6.2.4] (fp2) from rat liver microsomes. Both the generation of HA· and the oxidation of NADPH were increased with increasing the concentrations of alloxan or fp2. These results indicate that NADPH-linked HA·generation was catalyzed by fp2. The HA·was generated in the reaction of alloxan with a homogenate of rat islet cells. The generation of HA· in the reaction of alloxan with the homogenate of rat islet cells was markedly diminished by an immunogloblin fraction (Ig) of anti-fp2 serum, but not by control-Ig. The anti-fp2-Ig also inhibited the generation of HA· in the reaction system of alloxan with NADPH in the presence of fp2. When the homogenate of islet cells from phenobarbital-treated rats was used, the generation of HA· was signiflcantly increased in comparison with that from the control rats. However, the homogenate of islet cells from 3-methylcholanthrene-treated rats did not have such a stimulative effect. These results suggest that the reduction of alloxan to HA· was catalyzed by fp2 presented in islet cells.