A catalogue of the genes encoded by chromosome 21 would provide a framework for assigning roles in the etiology of Down syndrome (DS) to individual genes. We have begun generating such a catalogue, starting with a 1.2 Mb region surrounding the marker D21S55. Our efforts utilized the yeast artificial chromosome (YAC) and cosmidclone based high resolution physical maps that we have constructed of this region. Direct-selection of fetal brain cDNAs with YAC DNA was used to isolate transcribed sequences. The selected cDNA fragments were analyzed by limited DNA sequence analysis, Northern blot hybridization and screening of cDNA libraries. The cDNA fragments were assigned positions on the physical map by hybridization to a collection of cosmid clones. The accurate determination of map positions for individual cDNA fragments allowed us to determine sources of variability in the cDNA selection procedure. The combined analysis and mapping was used to estimate the completeness of our mapping efforts and to identify procedures that would facilitate large-scale transcript mapping. The transcribed sequence map that we have assembled will allow the importance to DS of genes in this region to be examined and will aid in the design of strategies for larger scale efforts. © 1994 Oxford University Press.