Loss of heterozygosity on human chromosome 16q is frequently observed in both ductal and lobular invasive breast carcinomas. We have generated a 2.8-megabase PAC contig covering the smallest region of loss of heterozygosity overlap on 16q22.1 (SRO2). We established the contig orientation with two-color fluorescence in situ hybridization and verified that the contig faithfully represents the SRO2 region using long-range mapping. We have identified 68 transcripts in the map on the basis of expressed sequence tag screening and CpG island subcloning. One of the genes residing within SRO2 is the E-cadherin gene, CDH1. This gene is known to be mutated in lobular breast carcinomas, resulting in loss of E-cadherin expression. However, E-cadherin shows normal expression in most cases of ductal carcinoma, the major mammary cancer type. Thus other genes within 16q22.1 are expected to be involved in the development of this tumor subtype. A minimal-tiling path of the contig presented consists of PAC clones, which have the potential of being transferred to mammalian cells as stably replicating episomes. This feature might serve as the basis for a functional strategy in which PACs would be introduced into tumor cells for the identification, verification and characterization of the tumor suppressor gene expected to be present within SRO2.