We sought to identify amplification target genes across the genome on the basis of a modification of the protocol developed by Pollack et al.1 for comparative genomic hybridization (CGH) analysis on complementary DNA microarrays. The same cDNA microarrays are then applied for expression analyses to identify directly genes whose expression is increased owing to amplification. We tested this approach by analyzing five breast cancer cell lines using a custom-made cDNA microarray specific for chromosome 17. The results were compared with data from conventional CGH analysis and indicated accurate detection of cell lines with HER-2 amplification (BT-474, SK-BR-3 and UACC-812) and those with amplification of the 17q23 region (BT-474 and MCF7). Compared with the exact copy number values available by fluorescence in situ hybridization, the CGH microarray analysis accurately defined the location and detailed structure of the amplicons along 17q23. Although we observed individual cDNA clones providing false-negative copy number estimates, averaging out signals over multiple adjacent clones provided accurate definition of all high-level amplifications. We observed numerous specific genes that showed both amplification and overexpression, such as HER-2, GRB7, MLN51 and MLN64 at17q12 as well as S6K and APPBP2 at 17q23. A few microarray experiments carried out within a two-week period allowed us to reproduce and extend the information obtained by conventional techniques over the course of the past few years. We are now carrying out CGH microarray analyses of these and other cell lines with an 8,000-clone array, which provides coverage of the entire genome at once.