Gene amplification and loss of heterozygosity are alterations to chromosomal structure whereby tumor cells alter patterns of gene expression. We have identified a novel mechanism of gene regulation in which cancer cells predominantly express one of the two alleles of a gene. Estrogen receptor (ER)-alpha is overexpressed in hormone-responsive breast cancer compared with normal breast epithelial cells. Using a polymorphism of codon 10, we examined allele-specific expression of the four different ER promoters in MCF-7 breast cancer cells and primary tumors. Monoallelic amplification of expression (MAX) for all four ER promoters was identified, resulting in an allelic preference of > 100-fold. MAX was the result of an amplification of allele copy number and a preference to transcribe the amplified allele. The effect of MAX was most significant for the promoters clustered near the 1' exon, whereas the expression from the distant H promoter mirrored template copy number. MAX of the ER gene was not found to occur in normal endometrial or breast tissue. As a novel mechanism in cancer genetics, MAX can result in functional homozygosity at a gene locus.