Amplification of 20q13.2 occurs in breast and other cancers and is associated with aggressive tumor behavior. We report the first sequence and comprehensive biological characterization of a tumor amplicon. Array-based comparative genomic hybridization resolved the 1.2-megabase amplicon into a pair of recurrent peaks. The proximal amplicon encodes the putative Zn-finger transcription factor ZNF217, a candidate oncogene recently shown to immortalize human mammary epithelial cells. Analysis of the genomic sequence for genes, repetitive elements, CpG islands, and gene expression revealed six previously discovered genes (ZNF217, ZNF218, PIC1-like, NABC1, CYP24, and NABC2) and four new genes (PFDN4, NABC3, NABC4 and NABC5). ZNF217 is the only protein-coding gene in the 160-Kb proximal amplicon peak. CYP24 and PFDN4 map in the distal amplicon. PFDN4 is overexpressed in tumors in which it is amplified and in some in which it is not. Amplicon breakpoints cluster in regions of very high repeat content flanking ZNF217 and PFDN4. A 14-Kb duplication, of a class associated with unstable chromosome regions, maps close to ZNF217. This duplicon is approximately 97% identical to a 14-Kb element on chromosome 22q13 and encodes one of three CpG islands in the amplicon and NABC3. We cloned and sequenced the syntenic region of mouse chromosome 2, revealing numerous homologies. These correspond to conserved exons and noncoding elements believed to be regulatory or structural in nature. We will report on these findings, which clearly demonstrate the power of comparative sequence analysis for cancer biology.