Amplification of the ERBB2 oncogene at 17q12 is clinically the most relevant genetic aberration in breast cancer and several studies have linked ERBB2 activation to poor clinical outcome. The development of targeted antibody-based therapy for ERBB2-overexpressing tumors and the possible role of ERBB2 as a predictor of chemotherapy treatment response have further emphasized the essential role of ERBB2 in breast cancer. Here, we performed a detailed characterization of the molecular events occurring at the ERBB2 amplicon in primary breast tumors. Analysis of the amplicon structure in 330 breast tumors by fluorescence in situ hybridization to a tissue microarray revealed a 280-kb common region of amplification that contains 10 transcribed sequences, including eight known genes. The expression levels of these 10 transcripts were determined in 36 frozen samples of grade-matched ERBB2-amplified and -nonamplified (as determined by fluorescence in situ hybridization) primary breast tumors by using quantitativereal-time reverse transcriptase-polymerase chain reaction. A highly significant association between amplification and expression levels was observed for six of these genes, including ERBB2 and two uncharacterized hypothetical proteins, MGC9753 and MGC14832. These results support the recent findings on the influence of copy number on gene expression levels and highlight novel genes that might contribute to the clinical behavior of ERBB2-amplified breast tumors.