In the human hepatitis B virus (HBV) genome, the 5' end of the polymerase coding sequence overlaps with the 3' end of the core protein coding sequence. Recent results obtained from genetic studies have suggested that translation of HBV polymerase initiates from the first ATG codon of the polymerase reading frame and is not a result of frameshift translation from the core protein reading frame, as in the case of retroviruses. By using in vitro-synthesized SP6 RNA transcripts, we now demonstrate that HBV core protein-specific mRNA can direct the synthesis of polymerase from the internal polymerase ATG codon in rabbit reticulocyte lysates and Xenopus oocytes. A related message with an additional 60 nucleotides at the 5' end (pre-core protein mRNA) was not as efficient as the core protein mRNA for translation of polymerase. Furthermore, translation of polymerase from the core protein mRNA was not inhibited by the cap analog m7GpppG. This result, together with the results described above, indicates that translation of HBV polymerase occurs in a novel, cap-independent manner.