We investigated whether replication-competent pre-C/C defective mutants of hepatitis B virus (HBV) are detectable in primary human hepatocellular carcinoma (HCC) tissues from patients of a geographic area endemic for such mutants. DNAs extracted from formalin-fixed paraffin-embedded HCC samples were checked for the presence of specific HBV DNA sequences using the polymerase chain reaction (PCR). Amplified pre-C regions from nine HCC samples were directly sequenced as were samples of nontumoral liver tissues from five of these patients. The data show that hypervariable distal pre-C sequences were present in all nine HCC samples; this high variability was dependent on point mutations, which led to amino acid substitutions in nearly all cases. Interestingly, seven of the nine HBV DNA-positive samples from HCC tissues (but not samples from peritumoral liver tissue) showed mutations leading to amino acid substitution at the level of a distal cysteine residue. No mutation generating a translationally defective pre-C/C region was detectable in the tumor samples. Otherwise, in four of the six nontumoral liver tissues available from the same patients, a pre-C sequence with an in-frame TAG stop codon was detectable, although in three cases as a component of mixed population.