Rodent-human somatic cell hybrids containing single human chromosomes or chromosome fragments are extremely valuable in physical mapping, marker analysis, and disease mapping. Chromosome 21 has been extensively studied in this fashion, and a single set of hybrids has been utilized in mapping the majority of chromosome 21 markers. The utility of a set of hybrids depends upon the definition of the human chromosome content. Recently, Chumakov and coworkers (1) utilized 198 chromosome 21 markers in the preliminary analysis of YACs spanning chromosome 21q. We have used these same markers to evaluate the STS content of a set of 27 chromosome 21 somatic cell hybrids, resulting in the description of the breakpoints at the molecular level, as well as the definition of 35 "bins. " The detailed molecular definition of chromosome 21 content of the hybrids, in combination with the further analysis of chromosome 21 YACs (2), has resulted in the most detailed picture of chromosome 21 to date.