We have studied the contribution of mismatch sequences to the trinucleotide repeat expansion that causes hereditary diseases. Using an oligonucleotide duplex, (CAG)5/(CTG)5, as a template-primer, DNA synthesis was carried out using either Escherichia coli DNA polymerase I (Klenow fragment) or human immunodeficiency virus type I reverse transcriptase (HIV-RT). Both enzymes expanded the repeat sequence longer than 27 nucleotides (nt), beyond the maximum length expected from the template size. The expansion was observed under conditions in which extension occurs either in both strands or in one strand. In contrast, with another template-primer that contains a non-repetitive flanking sequence 5'-upstream of the repetitive sequence, the reaction products were not extended beyond the template size (45 nt) by these DNA polymerases. We then used mismatched template-primers, in which either 1, 2 or 6 non-complementary nucleotides were introduced to the repeat sequence that is flanked by a non-repetitive sequence. In this case, primers were efficiently expanded over the expected length of 45 nt, in a mismatch-dependent manner. One of the primers with six mismatches extended as long as 72 nt. These results imply that the misincorporation of non-complementary deoxyribonucleoside monophosphates (dNMPs) into the repeat sequence makes double-stranded DNA unstable and triggers the slippage and expansion of trinucleotide repeats by forming loops or hairpin structures during DNA synthesis.