The X protein can act on the enhancer of hepatitis B virus in an in uitro system and elevate the transcriptional level of hepatitis B virus. However, because no relationship had been reported between X protein expression and hepatitis B virus replication in patients with chronic hepatitis B, we focused on its expression in the liver in comparison with markers of hepatitis B virus replication. Liver biopsy samples and sera from 59 carriers with HBsAg were examined immunohistochemically for X protein using rabbit IgG against recombinant X protein. There was a significant difference in the serum hepatitis B virus DNA level between X protein-positive and -negative patients (p < 0.001). Serum pre-S1 and pre-S2 antigens were also measured quantitatively by enzyme immunoassay using monoclonal antibodies specific against each antigen. The titers of pre-S1 antigen in patients positive for X protein were significantly higher (p < 0.001) than those of the X protein-negative patients (3.02 * 0.99 vs. 2.00 2 0.59, respectively). Similarly, the titers of pre-S2 antigen were 2.98 f 0.91 vs. 1.94 f 0.54, respectively (p < 0.001). The rate of positivity of the X protein was higher (38 of 49; 77.6%) in the replicative group (serum HBeAg, serum hepatitis B virus DNA or HBcAg in liver positive) compared with that observed in the nonreplicative group (3 of 10; 30% -serum HBeAg, serum hepatitis B virus DNA and HBcAg in liver negative) (p < 0.01). Our findings indicate that the X protein is closely correlated with hepatitis B virus replication and may have an important role in viral replication in chronic hepatitis B virus infection. (HEPATOLOGY 1991;13:417-421.) HBV contains at least four open-reading frames (ORF): S, C, P and X, and the functions of the former three have been clarified. As for the fourth ORF X gene, some in vitro studies suggest that the X protein acts on the enhancer and elevates the transcriptional level of HBV (1,2). However, the relationship between X protein and HBV replication is not clear in patients with HBV