Many eubacterial DNA polymerases are bifunctional molecules having both polymerization (P) and 5' nuclease (N) activities, which are contained in separable domains. We previously showed that the DNA polymerase I of Thermus aquaticus (TaqNP) endonucleolytically cleaves DNA substrates, releasing unpaired 5' arms of bifurcated duplexes. Here, we compare the substrate specificities of TaqNP and the isolated 5' nuclease domain of this enzyme, TaqN. Both enzymes are significantly activated by primer oligonucleotides that are hybridized to the 3' arm of the bifurcation; optimal stimulation requires overlap of the 3' terminal nucleotide of the primer with the terminal base pair of the duplex, but the terminal nucleotide need not hybridize to the complementary strand in the substrate. In the presence of Mn2+ ions, TaqN can cleave both RNA and circular DNA at structural bifurcations. Certain anti-TaqNP mAbs block cleavage by one or both enzymes, whereas others can stimulate cleavage of nonoptimal substrates.