Cu,Zn-superoxide dismutase was isolated from Aspergillus niger mycelia, harvested at the mid-logarithmic growth phase. The purification scheme aimed at the optimization of the ethanol/chloroform extraction (Tsuchihashi extraction) through response surface methodology. Upon optimum extraction conditions, it was possible to obtain electrophoretically pure enzyme preparations, by the application of one step anion exchange chromatography. The enzyme yield of this simple purification procedure was above 75% while the specific activity of the final preparation was among the highest reported for eucariotic microorganisms. The purified enzyme exhibited similar physicochemical characteristics with other Aspergillus sp. superoxide dismutases revealing an apparent tetrameric structure with a subunit molecular weight of 19 kDa, and a pl of 5.95.