We have resolved the sizes of the yeast artificial chromosomes (YACs) from an ordered library spanning the entire long arm of Chromosome (Chr) 21 to examine the proximity of sequence-tagged sites (STS) originally used to position these clones. The average insert length was 540 kilobases, and some 18% of the 765 clones have either lost or generated multiple YACs during cultivation. Comparing the sizes of YACs that share common sites allowed the identification of an additional 8% of the clones with large scale additions or deletions. Maximum physical distances between chromosome markers, as established by the co-occurrence of STS on a single YAC, generally agreed with those estimated by other procedures, except for a large region in 21q21. In addition to providing insights into the structure, mapping and organization of this chromosome, knowledge of the sizes and contents of these clones will greatly facilitate the acquisition of any sequence present in this library.