Deletion of genes from the chromosome 21 region between APP and SOD1 is a potential cause of some of the major phenotypic features of monosomy 21 patients. Fine physical mapping helps identify potential candidate genes. After selecting nonchimeric YACs by FISH analysis, we determined their marker contents by PCR and hybridization studies. Fifteen YACs were chosen and mapped by restriction enzyme analysis and labeling of end fragments. We localized 55 markers, including 31 STSs, 10 YAC ends, and 4 NotI linking clones, along a 6.7-Mb contig. This map facilitates transcriptional analysis of this region and construction of ready-to-sequence contigs. Furthermore, FISH mapping of two patients with partial monosomy 21 using YAC and cosmid clones allowed us to define more accurately the telomeric border of the critical region between markers S226 and S213.