A deletion of the long arm of chromosome 20, del(20q), is a recurring abnormality in malignant myeloid diseases. In previous studies, we delineated a commonly deleted segment (CDS) of 5 Mb within band 20q12 flanked by D20S206 (proximal) and D20S481 (distal). We have generated a detailed physical map of P1 artificial chromosome (PAC) clones of this interval as well as a transcriptional map. The contig consists of 81 clones to which 152 markers (27 genes, 45 unique expressed sequence tags (ESTs) or UniGenes, 24 polymorphisms, and 56 sequence-tagged sites) have been mapped. Using PAC clones for fluorescence in situ hybridization analysis of myeloid leukemia cells with reciprocal translocations of 20q, or unbalanced rearrangements leading to loss of 20q, we have narrowed the CDS to an approximately 250-kb interval encompassing two overlapping PACs, P201E16 and P29M7 (between EST AA368224 and D20S481). This interval is gene-rich and contains 5 characterized genes, 4 UniGenes, and 9 single ESTs. The development of a transcriptional map and the identification of the smallest CDS will facilitate the molecular cloning of a myeloid leukemia suppressor gene on 20q.