Abstract The goal of this study was to identify age-related changes in the expression of estrogen target genes in mouse uterus. We developed a novel ‘estrogen response element (ERE) Chip’ microarray bearing 297 genes including both known estrogen target genes and genes identified by searching the mouse genome database to have EREs, AP-1 sites, and Sp1 sites, all targets of estrogen receptor (ER) regulation. 400–500 bp PCR products of these 297 genes were printed onto nylon membranes creating the ‘ERE Chip’ microarray. This microarray is unique because it is the first estrogen-responsive gene-specific microarray to identify changes in uterine gene expression in young versus old mice. Using this ERE microarray we identified 10 uterine genes whose expression was up-regulated in old mice, e.g. β-actin, calcium binding protein 45a, Sp1, and COUP-TFII. In contrast, the expression of only 4 uterine genes, i.e., complement C3, lactoferrin, Muc-1, and 17-beta-hydroxysteroid dehydrogenase 8 (H2-Ke6) was down-regulated in old mice. These changes may reflect an increase in stromal and a decrease in glandular epithelial gene expression, and may be associated with age-related changes in these tissue compartments within the uterus, possibly leading to the decline in reproductive function in C57Bl/6 mice.