The tertiary structure of template-primer and gapped DNA substrates in the complex with rat polymerase β (pol β) has been examined using the fluorescence energy transfer method based on the multiple donor-acceptor approach. In these studies, we used DNA substrates labeled at the 5′ end of the template strand and the 5′ end of the primer with the fluorescent donor and/or acceptor. Measurements of the enzyme complex with the template-primer DNA substrate having a ten nucleotide long ssDNA extension indicate that the distance between the 5′ end of the template strand and the 5′ end of the primer decreases by ∼9.8 Å as compared to the free nucleic acid. Analogous experiments with the template-primer substrate, having the ssDNA extension with five nucleotide residues, show ∼6.6 Å distance decrease. Such large distance decreases indicate that the DNA is significantly bent in the binding site. Analysis of the data indicates that the bending occurs between the third and the fourth nucleotide of the ssDNA extension. The entire template strand is at the bend angle ΘTP = 85 ± 7° with respect to the dsDNA part of the DNA molecule. In the polymerase complex with the gapped DNA, the distance between the 5′ ends of the DNA and the bend angle are 66 ± 2.2 A° and 65 ± 6°, respectively. These values are very similar to the same distance and bend angle of the gap complex in the crystal structure of the co-complex. The presence of the 5′-terminal PO4- group downstream from the primer does not affect the tertiary conformation of the gapped DNA, indicating that the effect of the phosphate group is localized at the ssDNA gap.