Reverse transcription of the hepadnavirus genome initiates near the 3' end of the RNA template and has previously been shown to depend on sequences flanking the initiation site for DNA synthesis (C. Seeger and J. Maragos, J. Virol. 64:16-23, 1990). DNA synthesis leads to the covalent attachment of a protein to the 5' end of minus-strand DNA, and it is generally believed that this protein serves as the primer for reverse transcription. To examine priming in more detail, we have carried out a detailed genetic analysis of the nucleotide sequences at the origin of minus-strand DNA synthesis characterized in our earlier study. This mutational analysis has led to the identification of a short, four-nucleotide-long sequence as the signal for initiation of reverse transcription. This signal is a UUUC sequence motif flanking the position of the 5' end of minus-strand DNA, which alone is not sufficient for DNA synthesis, indicating that positional effects are also important to specify the origin of DNA synthesis.