Technical Abstract: Characterizing estrogen-responsive genes is an essential step towards fully understanding mechanisms of estrogen action during mammary gland development and function. To catalogue these genes, sixteen prepubertal heifers were used in a 2 x 2 factorial with ovarian status (intact or ovariectomized) as the first factor and estrogen (E) treatment as the second (excipient or estradiol). Heifers were ovariectomized at approximately 4.5 months of age and estrogen treatments were initiated one month later. After 3 days of treatment, gene expression in the parenchyma and fat pad of the bovine mammary gland was analyzed using a high-density oligonucleotide microarray. This microarray contained probes representing 40,808 Tentative Consensus sequences from the TIGR Bos taurus Gene Index and 4,575 singletons derived from libraries of pooled mammary gland and gut tissues. Microarray data were analyzed using the SAS Mixed Procedure with permutation testing. A total of 125 estrogen-responsive genes were identified using an experiment-wide permutation-based significance level of p < 0.1. Among these genes are known estrogen-targeted genes such as stanniocalcin 1, alpha-1-antiproteinase, progesterone receptor, nucleobindin 2, insulin-like growth factor 1, and tissue factor pathway inhibitor. However, the majority of the genes identified were not previously reported to be estrogen-responsive. In silico mapping and estrogen response element (ERE) search indicated potential EREs in the promoter regions of some of these novel estrogen-responsive genes. The distinctive expression patterns regulated by estrogen in parenchyma and fat pad suggest mechanisms of action and reciprocal signaling between cell types.