DNA polymerases catalyze the addition of deoxyribonucleotides onto DNA primers in a template-directed manner. The requirement for template instruction distinguishes these enzymes from other nucleotidyl transferases, such as terminal deoxynucleotidyl transferase, that do not utilize a template. An oligonucleotide substrate was used to characterize a novel, non-templated nucleotide addition reaction carried out by DNA polymerases from a variety of procaryotic and eucaryotic sources. The products of the reaction, in which a deoxyribonucleotide was added to the 3' hydroxyl terminus of a blunt-ended DNA substrate, were analyzed by electrophoresis on high resolution, denaturing polyacrylamide gels. DNA polymerase from Thermus aquaticus, polymerase alpha from chick embryo, rat polymerase beta, reverse transcriptase from avian myeloblastosis virus, and DNA polymerase I from Saccharomyces cerevisiae all carried out the blunt-end addition reaction. The reaction required a duplex DNA substrate but did not require coding information from the template strand. These results demonstrate that template instruction is not an absolute requirement for the catalysis of nucleotidyl transfer reactions by DNA polymerases.