The hepatitis B virus (HBV) DNA contains an open reading frame (ORF) designated X, which has the capacity to encode a protein of 16,560 Da (subtype adw). Such a protein has not been identified in either HBV particles or infected human livers, and therefore its role in the viral life cycle remains unknown. We report here the expression of the HBV X ORF in cultured cells using recombinant vectors. A protein of 16 kDa was identified by means of an antiserum prepared against a synthetic peptide and with human antisera from hepatitis B patients as well as those with hepatocellular carcinoma. Cell fractionation and immunofluorescence studies suggest a probable association with cytoskeletal components. Our studies further located a promoter sequence upstream of the X ORF, which directs the transcription of a 0.7- to 0.8-kilobase X-specific RNA in transfected human hepatoma cells.