A family of 340-bp tandem telomere-associated DNA repeats is present in 50- to 200-kb blocks in seven of the eight paired chromosome ends in Chironomus pallidivittatus. It consists of four main subfamilies, differing from each other by small clusters of mutations. This differentiation may reflect different functional roles for the repeats. Here we find that one subfamily, D3, is consistently localized most peripherally and extends close to the ends of the chromosomes, as shown by its sensitivity to the exonuclease Bal 31. The amounts of D3 are highly variable between individuals. The repeat characteristic for D3 forms a segment with pronounced dyad symmetry, which in single-strand form would give rise to a hairpin. Evidence from an interspecies comparison suggests that a similar structure is the result of selective forces. Another subfamily, M1, is present more proximally in a subgroup of telomeres characterized by a special kind of repeat variability. Thus, a complex block with three kinds of subfamilies may occupy different M1 telomeres depending on the stock of animals. We conclude that subfamilies are differentially distributed between and within telomeres and are likely to serve different functions.