FISH analyses and loss of heterozygosity studies have delineated a commonly deleted region in hematological malignancies flanked byETV6andCDKN1Bon chromosome 12p12.3. The same chromosomal region is also a target for deletions in certain solid tumors. As an initial step toward the cloning of a potential tumor suppressor gene at 12p12.3, we mapped theETV6–CDKN1Bregion physically using bacterial artificial chromosome (BAC) and P1-derived clone (PAC) contigs. The 1.2-Mb high-resolution, contiguous map extends from D12S1095 to D12S929 and consists of 19 PACs and 20 BACs. Pulsed-field gel electrophoresis experiments confirmed the integrity of the clone-based map and identified six CpG islands in the region. A transcript map was generated by performing hybridization selection experiments with the genomic clones, by evaluating known 12p ESTs for their presence in the contig, and by sequence analysis of CpG islands in the region. Altogether evidence was gathered for the presence of the recently publishedLRP6gene and at least seven other new genes in this chromosomal region. TheCLAPS3gene, mapped between D12S391 and D12S358, was reassigned to chromosome 5 since genomic sequencing demonstrated the chromosome 12p sequence to be a pseudogene. Polymorphic CA repeats were identified approximately every 100 kb, which will support future analysis of loss of heterozygosity in tumors. Fluorescencein situhybridization analysis of leukemia patients with del(12p) further refined the commonly deleted segment to 600 kb betweenETV6and D12S358, which apparently excludesCDKN1B.Methylation changes of the CpG islands in theETV6–CDKN1Binterval were assessed by Southern analysis for leukemia patients with hemizygous 12p deletions. A “de novo” methylation was detected only at theLRP6CpG island in 2 of 22 leukemia patients tested and was confirmed by methylation-sensitive PCR and sequencing. The genomic structure ofLRP6was elucidated to allow screening for inactivating mutations, but only intragenic polymorphisms were identified. Hypermethylation of CpG islands associated with gene promoters is reported as a common mechanism for gene silencing and tumor suppressor inactivation. Therefore the consequences of theLRP6CpG island methylation and its role in the observed phenotype need further investigation.