We investigated the generation of nitric oxide (NO) by H₂O₂dependent peroxidation of hydroxyurea in the presence of copper‐containing proteins such as Cu, Zn‐snperoxide dismutase (Cu, Zn‐SOD) or ceruloplasmin as a catalyst. In the reaction mixture of hydroxyurea, Cu, Zn‐SOD, and H₂O₂, “NO generation was identified by measuring the specific electron spin resonance (ESR) signal of 2‐phenyl‐4,4,5,5‐tetramethylimidazoline‐l‐oxyl 3‐oxide (PTIO). The ESR signal of the NO‐hemoglobin adduct was also detected in human red blood cells during copper‐catalyzed pcroxidation of hydroxyurea. The‘NO production during peroxidation of hydroxyurea was quantified as NO₂ formation, measured by using the Griess assay, and the amount of NO₂ was dependent on the concentration of hydroxyurea of the reaction mixture. ESR spin trapping with 5,5‐dimethyl‐l‐pyrroline JV‐oxide (DMPO) showed hydroxy radical COH) generation in the reaction of H₂O₂ with either Cu, Zn‐SOD or ceruloplasmin. Several “OH scavengers, such as ethanol, thiourea, DMPO, and dimethylsulfoxide, and the metalchelating agent diethylenetriaminepentaacetic acid significantly inhibited‘NO generation from hydroxyurea. This indicates that‘NO release from hydroxyurea may be mediated by‘OH derived from the copper‐catalyzed Fenton‐like reaction. Incubation of hydroxyurea and Cu, Zn‐SOD with xanthine oxidase and hypoxanthine in a system forming O₂→ H₂O₂ also resulted in appreciable‘NO production. These results suggest that‘NO production from hydroxyurea catalyzed by copper‐containing proteins may be the molecular basis of the pharmacological and antitumor action of hydroxyurea.