Chloroperoxidase (CPO) from Caldariomyces fumago is a highly versatile and synthetically important enzyme. Nevertheless, the actual technical application is limited, mainly due to the high costs for purified preparations. Here, a novel fast and very efficient method for the purification of CPO has been developed. It involves separation of CPO from black fungal pigment and most side proteins in aqueous biphasic systems followed by a single chromatographic step. A residual yield of similar to 70.4% and a specific activity of 2900 U/mg were obtained. Sufficient purity for technical application was already achieved after biphasic extraction, where CPO recovery from culture broth yielded about 100%. Time required for purification ranges between 30 min and 1 day depending on the desired degree of purity. Thus, a protocol is presented that reduces consumption of time and material and enhances the enzyme quality. (C) 2009 Elsevier Ltd. All rights reserved.