A method is described for purifying a collagenase fraction from commercial batches of the enzyme, which is free of proteolytic effects. The method, which is based on preparative electrophoresis in discontinuous buffers followed by electroelution, enables the separation and purification of 6 collagenase fractions with a good recovery of the protein (approximately 80%). Proteinase activity was a peculiarity of the low molecular weight components whereas one high MW fraction (C2) had maximal collagenase activity but was free from aspecific proteolytic effects. Only this collagenase should be employed for molecular studies on the collagen composition of the basement membrane.