A partial purification of epidermal G2 chalone from crude epidermal cell extracts from hairless mice is described. The procedure involved the sequential use of ammonium sulphate precipitation, affinity chromatography and gel filtration. Mitosis-inhibiting activity at each stage in the purification was tested in an in vitro assay system employing human epidermoid carcinoma cells in exponential growth phase and Colcemid (Ciba) for arresting mitoses. This assay system is much more rapid and convenient than conventional in vivo systems. By the procedure described, a 10,000-fold material purification of the active component has been obtained. This corresponds to a 3,000-fold purification measured by protein content, and a 300-fold increase in mitosis-inhibiting activity per unit we;ght. The active component is acidic, contains sugar residues and has gel chromatographic properties characteristic of a substance with molecular weight of approximately 20,000. On SDS polyacrylamide gel electrophoresis, however, three weak bands were found. The active component is resistant to trypsin and protease and stable between pH 6.0 and 8.5. It is easily inactivated at pH values below 6.0. At the present stage of purification, several components other than the active one still remain in our material and further purification steps must be eventually employed.