To establish an in vivo experimental system for developmental endocrinology research, AtT-20 cells, a corticotropic tumor cell line, were transplanted by exo utero manipulation into mouse embryos on embryonic day 14. The induced tumor secreted ACTH in situ, and the circulating ACTH level was elevated. This was the first model for studying the regulation of ACTH in the mouse fetal adrenal in vivo and the first continuous ACTH treatment model in rodent fetuses. The changes in the adrenal gland from the tumor-induced embryos were analyzed by light microscopic morphometry, immunohistochemistry for steroidogenic enzymes, and electron microscopy. In the treated adrenal, the volume of the inner cortical zone was significantly larger than that in controls. In the inner zone, cell density was decreased, and average cell size was increased, whereas bromodeoxyuridine-incorporation was not increased. The enlarged inner zone cells expressed an enhanced level of cytochrome P45011beta, the corticosterone-synthesizing enzyme, and the serum corticosterone level was increased. Electron microscopy showed an active form of the organelles involved in steroidogenesis. These findings indicate that ACTH stimulates both adrenocortical hypertrophy and steroidogenesis in fetal mice. Potential perspectives of the novel paradigm in this research for molecular developmental endocrine study are discussed.