The tandemly repeated 28-bp sequence in the 5'-terminal regulatory region of human thymidylate synthase (TSER), which has been reported to be polymorphic in different populations, was surveyed in 668 Chinese from 9 Han groups, 8 ethnic populations, and 36 individuals representing a three-generation pedigree. Amplified fragments were separated by electrophoresis on 4% agarose gel. In addition to the reported double and triple repeats of the 28-bp sequence in TSER, we also detected a novel quintuple repeat in this region. The transient expression activity of TSER with the quintuple repeat is almost the same as that of the reported TSER with the triple repeat. All three alleles of the repeat type (2, 3, and 5) were further confined by sequencing. The frequencies of the TSER allele 2 and 3 were 18.82 and 81% in totally unrelated Chinese samples, respectively, while the frequency of allele 3 was variable in different Chinese populations with a range from 62 to 95%. On the basis of the sequences of the different alleles, the existence of the tandem repeats in each allele might be explained by slipped-strand mispairing during DNA replication.