Identification of estrogen-responsive genes is an essential step towards understanding mechanisms of estrogen action during mammary gland development. To identify these genes, sixteen prepubertal heifers were used in a 2 x 2 factorial experiment, with ovarian status (intact or ovariectomized) as the first factor and estrogen treatment as the second (control 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 was analyzed in the parenchyma and fat pad of the bovine mammary gland using a high-density oligonucleotide microarray. Oligonucelotide probes represented 40,808 Tentative Consensus sequences from the TIGR Bos taurus Gene Index and 4,575 singleton expressed sequence tags derived from libraries of pooled mammary gland and gut tissues. Microarray data were analyzed using the SAS Mixed Procedure with an experiment-wide permutation-based significance level of P < 0.1. Considerable differences in basal gene expression were noted between mammary parenchyma and fat pad. A total of 124 estrogen-responsive genes were identified, with most responding only in the parenchyma or the fat pad. The majority of genes identified were not previously reported to be estrogen-responsive. These undoubtedly include genes that are regulated indirectly, but also include known estrogen-targeted genes, and novel genes with potential estrogen-responsive elements in their promoter regions. The distinctive expression patterns regulated by estrogen in parenchyma and fat pad shed light on the need for both tissues to obtain normal mammary development.