Abstract: The subtelomeric Y' family of repeated DNA sequences in the yeast Saccharomyces cerevisiae is of unknown origin and function. Y's vary in copy number and location among strains. Eight Y's, from two strains, were cloned and sequenced over the same 3.2-kb interval in order to assess the within- and between-strain variation as well as address their origin and function. One entire Y' sequence was reconstructed from two clones presented here and a previously sequenced 833-bp region. It contains two large overlapping open reading frames (ORFs). The putative protein sequences have no strong homologies to any known proteins except for one region that has 27% identity with RNA helicases. RNA homologous to each ORF was detected. Comparison of the sequences revealed that the known long (Y'-L) and short (Y'-S) size classes, which coexist within cells, differ by several insertions and/or deletions within this region. The Y'-Ls from strain Y55 also differ from those of strain YP1 by several short deletions in the same region. Most of these deletions appear to have occurred between short (2-10 bp) direct repeats. The single base pair polymorphisms and the deletions are clustered in the first half of the interval compared. There is 0.30-1.13% divergence among Y'-Ls within a strain and 1.15-1.75% divergence between strains in the interval. This is similar to known unique sequence variation but contrasts with the 8-18% divergence among the adjacent subtelomeric repeats, X. Subsets of Y's exhibit concerted evolution; however, more than one variant appears to be maintained within strains. The observed sequence variation disrupts the first ORF in many Y's while most of the second ORF including the putative helicase region is unaffected. The structure and distribution of the Y' elements are consistent with having originated as a mobile element. However, they now appear to move via recombination. Recombination can account for the homogenization within subsets of Y's but does not account for the maintenance of different variants.