The 3' leads to 5' deoxyribonuclease activity associated with an Ustilago maydis DNA polymerase hydrolysed non-complementary 3'-primer termini about 12 times more rapidly than complementary termini. An analysis of its substrate specificity suggested that, although it was unable to hydrolyse fully single-stranded polynucleotides, it could hydrolyse such regions less than about four nucleotides in length covalently bound to a primer molecule which was base-paired to a complementary template strand. Template-primer combinations containing complementary or non-complementary primer termini both supported polynucleotide synthesis, but whereas the former were conserved, the latter were hydrolysed during the reaction thus allowing synthesis to occur. No addition of nucleotides onto a conserved non-complementary 3'-primer terminus was detected. The deoxyribonuclease activity therefore fulfilled a proof-reading function during DNA synthesis in vitro.