In breast cancer, several chromosomal sites frequently undergo amplification, implicating the location of genes important for tumor development and progression. Here we cloned two novel genes, breast carcinoma amplified sequence 3 (BCAS3) and 4 (BCAS4), from the two most common amplification sites in breast cancer, 17q23 and 20q13. The BCAS3 gene at 17q23 spans more than 600 kb at the genomic level and was predicted to encode a 913 amino acid nuclear protein. The BCAS4 gene at 20q13.2 encodes a 211 amino acid cytoplasmic protein. Both BCAS3 and BCAS4 represent novel genes with no homologies to any other known gene or protein. In the MCF7 breast cancer cell line, the BCAS3 and BCAS4 genes were co-amplified, and cloning of a highly overexpressed 1.3-kb transcript revealed a rearrangement fusing the last two exons of BCAS3 with BCAS4. The fusion led to a novel message in which only the first exon of BCAS4 and part of exon 23 of BCAS3 were transcribed. The BCAS4–BCAS3 fusion transcript was detected only in MCF7 cells, but the BCAS4 gene was also overexpressed in nine of 13 breast cancer cell lines. In conclusion, our results indicate that these novel genes, BCAS3 at 17q23 and BCAS4 at 20q13.2, undergo amplification, overexpression, and fusion in breast cancer and therefore may have a role in the frequent chromosomal alterations affecting these two loci. Published 2002 Wiley-Liss, Inc.