According to current knowledge, DNA polymerases accommodate only two polynucleotide strands in their catalytic site: the template and the primer to be elongated. Here we show that in addition to these two polynucleotide strands, HIV-1 and AMV reverse transcriptases, human DNA polymerases beta, gamma, and lambda, and the archaebacterial Dpo4 can elongate 10-nucleotide primers bound in a triple-helix manner to hairpin duplex DNA tethered by a few thymidine residues. The elongation occurs when the primer is parallel to the homologous strand. This feature was confirmed by using complementary single-stranded DNA with restricted nucleotide composition which bound polypurine and polypyrimidine primers at an asymmetric site. The results unambiguously confirmed the previous experiments, showing binding of the primer strand parallel to the homologous sequence. The common feature of these DNA polymerases is that they all elongated dG-rich primers, whereas they behaved differently when other polynucleotide sequences were used. Interestingly, only five to seven dG residues at similar positions between the primer and its binding site can allow elongation, which may even be facilitated by a single C/C mismatch. We suggest that DNA polymerases displace the primer form Hoogsteen bonds to from Watson-Crick pairings, enabling subsequent priming of replication. These experiments indicate that DNA polymerases may bind three DNA strands, as RNA polymerases do, and provide a molecular basis for 3'-OH invasion at short similar sequences in the DNA double helix, yielding potential DNA rearrangements upon single-strand breakage.