The genome of the hepatitis B virus contains a sequence (X gene) whose role is unclear. The almost complete region of the hepatitis B virus X gene was expressed in Escherichia coli, with the resulting protein being approximately 17 kilodaltons in molecular weight. Sera from 139 subjects were analyzed by Western blot analysis. Of the hepatitis B surface antigen-positive patients, anti-X was not found in 4 patients with acute hepatitis and in 12 healthy carriers, but was present in 41% (21/51) of the patients with chronic hepatitis, 63% (15/24) of those with liver cirrhosis, and 46% (12/26) of those with hepatocellular carcinoma. The expression of the X product in the liver tissues (43 hepatitis B surface antigen-positive patients) was investigated using an indirect immunohistochemical method. The X protein was observed in 64% (21/33) of the patients with chronic hepatitis and 50% (5/10) of those with liver cirrhosis, and was found when the serum was negative for anti-X. Hepatitis B core antigen was frequently expressed together with the X protein in the liver. The conclusions reached were that the frequency of anti-X increases with the length of chronic hepatitis B virus infection, that anti-X may suppress the expression of the X protein in the liver, and that the X protein may be related to hepatitis B virus replication.