Previous reports from this laboratory have indicated that a number of cytosol and nuclear proteins of Novikoff hepatoma cells were immunologically related [Yeoman, L. C., Jordan, J. J., Busch, R. K., Taylor, C. W., Savage, H., & Busch, H. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 3258; Busch, R. K., & Busch, H. (1977) Tumori 63, 347]. In preparation for analysis of their structure and function, studies were undertaken to purify nuclear antigen 2 from the cytosol of Novikoff hepatoma cells in high yield and purity. It was shown on Ouchterlony gels that cytosol nuclear antigen 2 formed a single immunoprecipitin band of identity with one of the bands extracted from Novikoff nuclear chromatin. In this study, a 70 000 molecular weight antigen was isolated from the cytosol of Novikoff hepatoma cells by ammonium sulfate fractionation, ion-exchange chromatography, and isoelectric focusing in a granulated gel bed. This protein which focused at a pI of 6.3 was labeled with 125I-labeled Bolton-Hunter reagent and purified on an Ultrogel AcA-44 column. As shown by electrophoresis on NaDodSO4-polyacrylamide gels, the antigen in the excluded volume migrated as a single protein with a molecular weight of 70 000. The overall purification over the starting material was 2890-fold.