To explore the role of hepatitis B virus (HBV) X protein in liver carcinogenesis, independently from its role in viral replication, we have analyzed X gene structure and expression in tumorous and non-tumorous tissues obtained from 9 hepatitis B surface antigen (HBsAg)-negative, HBV DNA-positive patients. HBV replication was undetectable in tumorous tissues. HBV X gene was truncated at its 3' end in 5 of 9 tumorous tissues and 1 of 8 non-tumorous livers. Sequence analysis performed on uninterrupted X genes from 3 tumors and 3 surrounding non-tumorous tissues showed a high rate of mutations, selectively in the tumorous livers. In 1 of the 3 tumors, a frameshift mutation induced a new stop at codon 129. HBV RNAs were tested by reverse transcriptase-polymerase chain reaction (RT-PCR) with surface (S), core (C) and X specific primers. X, but not S and C, RNA expression was found in 6 of 8 tumors and in 6 of 7 non-tumorous tissues. This finding was consistent with immunohistochemical detection of X, but not S and C, antigens in all tumors also expressing X RNA. Our results provide evidence for selective expression of HBV X, but not S and C, RNA and protein in the tumorous and non-tumorous tissue of HBsAg-negative, HBV DNA-positive patients. It also shows that the structure of the X gene is modified (interrupted or highly mutated) in the majority of tumorous livers. Taken together, our findings are consistent with a potential role of mutated X proteins in HBV-related liver oncogenesis.