Chromosome 21 is a model for the study of human chromosomal aneuploidy, and the construction of its physical and transcriptional maps is a necessary step in understanding the molecular basis of aneuploidy-dependent phenotypes. To identify the gene(s) responsible for Down syndrome congenital heart disease (DS-CHD), we constructed a physical map of the D21S55 to MX1 region. A bacterial artificial chromosome (BAC) library was screened using several YACs spanning the interval, and a P1-derived artificial chromosome (PAC) library was screened using radiolabeled STS PCR products and whole BACs in gap-filling initiatives. FISH confirmed the location of all BAC and PAC clones to 21q22.2-q22.3. Overlaps were established using clone-to-clone Southerns and 24 new STSs, generated from the direct sequencing of BAC and PAC ends, along with 35 preexisting STSs. Approximately 3.5 Mb of the 4- to 5-Mb D21S55 to MX1 interval is covered in 85 BACs and 24 PACs, representing fourfold coverage within the contigs. These BAC and PAC contigs are valuable reagents for isolating the genes for DS-CHD.