We have assigned six polymorphic DNA segments to chromosomal subregions and have established the physical order of these sequences on the long arm of chromosome 21 by in situ hybridization of cloned probes to normal metaphase chromosomes and chromosomes 21 from individuals with three different structural rearrangements: an interstitial deletion, a ring chromosome, and a reciprocal translocation involving four different breakpoints in band 21q22. Segments D21S1 and D21S11 map to region 21q11.2----q21, D21S8 to 21q21.1----q22.11, and D21S54 to 21q21.3----q22.11; D21S23 and D21S25 are both in the terminal subband 21q22.3, but they are separated by a chromosomal breakpoint in a ring 21 chromosome, a finding that places D21S23 proximal to D21S25. The physical map order D21S1/D21S11-D21S8-D21S54-D21S23-D21S25 agrees with the linkage map, but genetic distances are disproportionately larger toward the distal end of 21q.