Comparative genomic hybridization studies indicate that the 17q23 region is one of the most common amplification sites in breast cancer and therefore that it may harbor genes playing an important role in tumor progression. We have used a combination of molecular, genomic and microarray technologies to identify systematically target genes for this amplicon. We constructed a genomic contig for the 17q23 region (approximately 4 megabases) and used these genomic clones to map amplicon boundaries in 5 breast cancer cell lines and 372 primary breast cancers; 17 known genes and 26 expressed sequence tags were localized to the contig. Analysis of the genomic sequence of BAC clones from this region revealed 34 additional Unigene clusters and 43 individual expressed sequence tags. To perform a complete expression survey, we constructed a complementary DNA microarray containing 116 clones from our contig and 522 clones from other regions on chromosome 17. Microarray analysis on eight breast cancer cell lines revealed a limited number of consistently overexpressed genes, including S6K, RAD51C and APPBP2, and several unknown expressed sequence tags. The S6K coding for a regulator of the G1–S transition showed high-level overexpression by northern and western blot analysis as well as increased functional activity when amplified in breast cancer cell lines. S6K was amplified in 8.8% and overexpressed in 15% of primary breast tumors and showed a significant association with poor prognosis (P<0.002). Comprehensive analysis of the 17q23 amplicon revealed genes that may have oncogenic potential and may contribute to the more aggressive clinical course in breast cancer patients with 17q23-amplified tumors.