A commercial chromatofocusing system was applied to Cohn's fraction III of human serum to purify hyaluronidase (E.C.3.2.1.3.5). The protein that eluted in the pH range 4.7-5.3 was pooled and precipitated by adding ammonium sulphate to 50% saturation. This sequence of fractionation purified hyaluronidase extensively by immunological criteria. It is shown that hyaluronidase is a population of enzymes displaying microheterogeneity. The commercial chromatofocusing system behaved as theoretically expected. The capacity of the gel is 3 mg per ml gel. Any overload will be trapped or precipitated in the gel. The gel is easy to handle and did not deteriorate on repeated use.