To identify cell populations directly responsive to prolactin (PRL), growth hormone (GH), erythropoietin (EPO), or granulocyte-colony stimulating factor (GCSF) within the physiological setting of an intact mammal, we combined in situ detection of hormone-activated Stat5 in rats with high throughput tissue array analysis using cutting edge matrix assembly (CEMA). Inducible activation of Stat5a/b, as judged by levels of nuclear-localized, phosphoTyr694/699-Stat5a/b, served as an immediate and sensitive in situ marker of receptor signaling in rat tissues after injection into male and female rats of a single, receptor-saturating dose of hormone for maximal receptor activation. CEMA tissue arrays facilitated analysis of most tissues, including architecturally complex, thin-walled and stratified tissues such as gut and skin. In 40 tissues analyzed, 35 PRL-responsive and 32 GH-responsive cell types were detected, of which 22 cell types were responsive to both hormones. Interestingly, PRL but not GH activated Stat5 in nearly all of the endocrine glands. In mammary glands, PRL activated Stat5 in a majority of luminal epithelial cells but not in myoepithelial cells, stromal fibroblasts, or adipocytes, whereas GH activated Stat5 in a significant fraction of myoepithelial cells, fibroblasts, and adipocytes but only in a minority of luminal cells. Finally, the organism-wide screening revealed a yet-to-be identified EPO-responsive cell type in connective tissue. CEMA tissue arrays provide cost-effective in situ analysis of large numbers of tissues. Biomarker-based identification of cell populations responsive to individual hormones may shed new light on endocrine disease, as well as improve understanding of effects and side-effects of hormones and drugs.