Highly purified hepatitis B virus core particles were obtained in large amounts from the cytoplasm of infected human liver cells. This DNA polymerase-negative core preparation had only hepatitis B core antigen-specific antigenicity and showed a surprising stability. Two forms of a single protein of 22,000 molecular weight, P22, were resolved electrophoretically; the slower moving species, P22a, appeared to be a reduced form of the protein, and the faster moving species, P22b, could have represented a conformational isomer containing an intramolecular disulfide bond(s). The immunological properties and DNA-binding activity of the reduced form, P22a, were examined following separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by transfer onto nitrocellulose membranes (Western blotting). We found that the hepatitis B virus C gene protein shared the antigenic site responsible for both hepatitis B core and e antigen reactivity. We also demonstrated that the core protein(s) bound specifically the genomic hepatitis B virus DNA in comparison with a plasmid DNA (pBR322). This last observation was further substantiated by a radioimmunological method. P22a was also found to be phosphorylated in vitro by the endogenous protein kinase activity, copurified with the hepatitis B core antigen particles. These findings suggest that P22 is a multifunctional protein which is incorporated into core particles within the cytoplasm of the host cell before DNA encapsidation. A critical role of this protein in hepatitis B virus assembly is suggested.