As part of an effort to isolate genes from the entire long arm of human chromosome 21, we performed cDNA selection with 15 YACs from the regions of the pericentromeric heterochromatin and the two Giemsa dark bands of this chromosome using cDNA libraries from six different tissues. Nine of these YACs mapped to the Giemsa dark band, 21q21. The 9 YACs cover approximately 6 Mb of DNA, representing 15% of 21q and a significant portion of the 12-15 Mb estimated to be within this band. Several lines of evidence from analysis of the selected cDNA libraries suggest that this region of 21q has very few single-copy transcribed sequences. An EcoRI library was constructed with DNA from 1 of the 9 YACs. Grail analysis of the sequences of both ends of 24 YAC-specific clones from this EcoRI library revealed no potential exons. In contrast to these results, the selected cDNA libraries of a control YAC from the human MHC region in 6p21.3 as well as those from most of the other 21q YACs consisted largely of YAC-specific single-copy cDNA clones. Given the success of the cDNA selection method for finding a large number of genes in YACs from other chromosomal regions, these results suggest that the 6 Mb of DNA in the dark band 21q21 contains few single-copy sequences expressed in this tissue set. In contrast, selected cDNA libraries from the pericentromeric region, the telomeric border of the dark band 21q21, and the dark band 21q22.2 yielded more than 30 new ESTs.(ABSTRACT TRUNCATED AT 250 WORDS)