Computer analysis of DNA polymerase protein sequences revealed previously unidentified conserved domains that belong to two distinct superfamilies of phosphoesterases. The alpha subunits of bacterial DNA polymerase III and two distinct family X DNA polymerases are shown to contain an N-terminal domain that defines a novel enzymatic superfamily, designated PHP, after polymerase and histidinol phosphatase. The predicted catalytic site of the PHP superfamily consists of four motifs containing conserved histidine residues that are likely to be involved in metal-dependent catalysis of phosphoester bond hydrolysis. The PHP domain is highly conserved in all bacterial polymerase III alpha subunits, but in proteobacteria and mycoplasmas, the conserved motifs are distorted, suggesting a loss of the enzymatic activity. Another conserved domain, found in the small subunits of archaeal DNA polymerase II and eukaryotic DNA polymerases alpha and delta, is shown to belong to the superfamily of calcineurin-like phospho-esterases, which unites a variety of phosphatases and nucleases. The conserved motifs required for phospho-esterase activity are intact in the archaeal DNA polymerase subunits, but are disrupted in their eukaryotic orthologs. A hypothesis is proposed that bacterial and archaeal replicative DNA polymerases possess intrinsic phosphatase activity that hydrolyzes the pyrophosphate released during nucleotide polymerization. As proposed previously, pyrophosphate hydrolysis may be necessary to drive the polymerization reaction forward. The phosphoesterase domains with disrupted catalytic motifs may assume an allosteric, regulatory function and/or bind other subunits of DNA polymerase holoenzymes. In these cases, the pyrophosphate may be hydrolyzed by a stand-alone phosphatase, and candidates for such a role were identified among bacterial PHP superfamily members.