We studied recognition and binding of synthetic template-primers by Drosophila DNA polymerase alpha (pol alpha) holoenzyme. The template-primers used contained either mismatched base pairs at various positions in the primer region or exocyclic propanodeoxyguanosine (PdG) adducts at various positions in both template and primer.pol alpha requires primer-terminal complementarity of greater than or equal to 4 base pairs for efficient binding and incorporation. When a mismatched base pair is at the -4 position relative to the 3'-primer terminus, minimal but detectable binding occurs. This is consistent with the ability of pol alpha to incorporate a single nucleotide on a template-primer containing a mismatch at this position, but at a rate of only 7% relative to incorporation on a perfectly matched template-primer. No binding or incorporation (less than 1% of incorporation on a perfectly matched template-primer) was evident when a mismatched base pair was at the -3 position or closer, relative to the 3'-primer terminus. Similar results were obtained when PdG was placed at various positions in the primer region. When a PdG residue was located in the template region (+ 3 position relative to the 3'-primer terminus), single-nucleotide incorporation was stimulated 3-4-fold. These observations suggest that there are intrinsic aspects to the mechanism of nucleotide incorporation by pol alpha which ensure the fidelity of DNA synthesis by this enzyme and may provide novel insights into the fundamental mechanism of polymerase translocation along templates.