A novel hepatitis B viral (HBV) protein of 35-40 kDa, characterized by antibodies and proposed as an X-C fusion protein, was previously described in core particles isolated from HBV-, WHV-, and GSHV-infected livers. The X and C genes are two adjacent genes in all mammalian hepadnaviruses but are not contiguous in WHV and GSHV. After examination of the X and preC/C junction sequences of 10 HBV, 4 WHV, and 1 GSHV, we found that the ORF of preC can be extended 7 more sense codons upstream so that X overlaps with the preC/C gene in all sequences. The number of overlapping base pairs (bp) is varied: 46 by in HBV, 19 by in WHV, and 10 by in GSHV. In this region a conserved Atrack was found to be followed by a pair of inverted repeats, suggesting that a ribosomal frameshift may occur for X-C fusion protein production. To assess this possibility, we have used an in vitro transcription and translation coupling system to identify X-C protein production. Two recombinant SP6 plasmids were used. One contained a full length of the X and preC/C gene of wild-type HBV-DNA and the other fused the X-preC/C gene by inserting a 10-bp HindIII linker at the junction of the X-preC/C region. No X-C fusion protein was detected from the wild-type plasmid. In contrast a large amount of X-C fusion protein was produced from the linker-inserted clone. It appears, therefore, that the X-C fusion protein is unlikely to be produced via the mechanism of ribosomal frameshifting.