The gene-rich telomeric region of 21q harbors several loci relevant to human diseases including autoimmune polyglandular disease type I, nonsyndromic deafness, Knobloch syndrome, holoprosencephaly, and bipolar affective disorder. A contig of genomic clones in this region would facilitate the isolation of these genes. However, distal 21q22.3 has yet been poorly mapped, presumably due to the presence of sequences that are underrepresented in yeast artificial chromosome (YAC) libraries. We generated a framework of YACs and used these clones as starting points for the isolation of a combination of bacterial artificial chromosome clones, P1-derived artificial chromosome clones, and cosmid clones by chromosome walking procedures. These studies resulted in the construction of a high-resolution contig map spanning the 2.5-Mb region from PFKL to the telomere, approximately 2 Mb of which are covered by ready-to-sequence contigs. Within this map we determined the location and relative distance of 21 markers. These include 9 established genetic markers, the order of which is cen-PFKL-D21S154-D21S170-D21S171-D21S1903- D21S1897- D21S112-D21S1446-D21S1575-tel. Moreover, we established the precise map position of 13 genes and 4 ESTs including the recently isolated genes C21ORF2, SMT3H1, RNA editing deaminase 1 (ADARB1), folate transporter (SLC19A1), COL18A1, lanosterol synthase (LSS-PEN), pericentrin (PCNT), and arginine methyltransferase (HRMT1L1). This integrated map provides a useful resource for the mapping and isolation of disease genes and for the construction of a complete transcription map of distal 21q as well as for large-scale sequencing efforts.