Phenylglyoxal, an arginine-specific reagent, strongly inhibits DNA polymerases isolated from eukaryotic, prokaryotic, and RNA tumor viral sources as well as Escherichia coli RNA polymerase. The inhibitory action of phenylglyoxal appears to be due to interference with the template binding function of these enzymes and implies the presence of an arginine residue at the template binding site of these enzymes from diverse sources and suggests that template dependent DNA, and perhaps RNA polymerases, may be mechanistically similar with respect to their template binding function. In contrast, the activity of terminal deoxynucleotidyl-transferase, a template-independent DNA polymerase isolated from calf thymus, is not inhibited by phenylglyoxal. A detailed analysis of the inhibitory process carried out using avian myeloblastosis virus (AMV) DNA polymerase as a test enzyme revealed that inclusion of template-primer during the preincubation with phenylglyoxal, but not substrate triphosphates or primer alone, protects the enzyme against phenylglyoxal inactivation. Furthermore, phenylglyoxal does not appear to inhibit the elongation of initiated DNA strands, but blocks the reinitiation of DNA synthesis.