V chromosome 21 (ch. 21) flow-sorted library was screened for the presence of unique DNA segments which are specific for the 21st chromosome. By combining the techniques of somatic cell genetics and in situ hybridization, we have identified several of these recombinant probes and have regionally mapped one of them to the distal half of the long arm of chromosome 21 (q22.1- greater than qter). This represents the first report of the sublocalization of a unique DNA segment to chromosome 21 by in situ hybridization.