Cytogenetic analyses were performed on 223 breast carcinomas, of which 60% contained homogeneously staining regions (hsr), an intrachromosomal cytogenetic feature of gene amplification. The precise hsr localization could be determined for 123 hsr from 72 cases. The juxtacentromeric region of chromosome 8, band 11q13, and the whole of chromosome 17 were frequently involved. For 28 cases, the origin of the DNA sequences forming HSR could be investigated by chromosome painting, comparative genomic hybridization, and/or Southern blotting. Sequences from chromosomes 11 and 17 were mostly found within hsr located on chromosomes 11 and 17, respectively. In contrast, sequences from chromosome 8 were rarely found within hsr localized on chromosome 8. These observations suggest that different mechanisms lead to hsr formation in breast cancer. Band 11 q13 and the 17p chromosome arm may correspond to sites of in situ amplification driven by deletions distal to the amplification target genes. hsr in the region 17q2, which is also a frequent site of in situ amplification, takes place without the occurrence of a distal deletion. The short arm of chromosome 8 is often deleted, but frequently becomes the site of hsr formed elsewhere in the genome.