Intracellular β-galactosidase from Penicillium chrysogenum NCAIM 00237 was purified by procedures including precipitation with ammonium sulfate, ion-exchange chromatography on DEAE–Sephadex, affinity chromatography, and chromatofocusing. These steps resulted a purification of 66-fold, a yield of about 8%, and a specific activity of 5.84 U mg −1 protein. Some enzyme characteristics were determined using o-nitrophenyl-β-d-galactopyranoside as substrate. The pH and temperature optimum of the activity were about 4.0 and 30°C respectively. The K m and pI values were 1.81 mM and 4.6. β-Galactosidase of P. chrysogenum is a multimeric enzyme of about 270 kDa composed of monomers with a molecular mass of 66 kDa.