Amplification of the chromosome 20q13 region was recently discovered in breast cancer by comparative genomic hybridization and subsequently further defined by fluorescence in situ hybridization with specific probes. The target gene of the amplification remains unknown. Here, fluorescence in situ hybridization with a cosmid probe for the minimal region of amplification (RMC20C001) was used to study 20q13 amplification in 132 primary breast carcinomas and 11 metastases. The size of the amplicon was studied with four flanking probes. Thirty-eight (29%) primary tumors and 3 (27%) metastases showed increased copy number of the RMC20C001 probe (>1.5-fold relative to the p-arm control). Nine (6.8%) of the primary tumors were highly (>3-fold) amplified. Although the size and location of the amplified region varied from one tumor to another, only the RMC20C001 probe was consistently amplified. 20q13 amplification was significantly associated with a high histological grade (P = 0.01), DNA aneuploidy (P = 0.01), and high S-phase fraction (P = 0.0085). High-level amplification was also associated with short disease-free survival of patients with node-negative breast cancer (P = 0.002). We conclude that high-level 20q13 amplification may be an indicator of poor clinical outcome in node-negative breast cancer and that this chromosomal region is likely to contain a gene with an important role in breast cancer progression. A large definitive study is warranted to assess the independent prognostic value of 20q13 amplification.