Rat endometrial cells were isolated from ovariectomized adults submitted to a progesterone-estradiol sequence proper to sensitize the uterus to respond to a deciduogenic stimulus, and then cultured in vitro in the presence of progesterone. They grew rapidly and became morphologically similar to the decidual cells occurring in vivo with regard to the binucleation and the accumulation of characteristic filamentous material in the cytoplasm. The life-span of the decidualized cells in vitro was comparable to the duration of the deciduoma in vivo. The progesterone treatment of the rat was found to be a prerequisite for such development and transformation of endometrial cells in vitro, since control experiments performed with nonhormonally injected castrated animals gave only isolated islets of epithelial cells and scattered fibroblast-like cells in culture, whereas the decidual transformation was still observed, although with some delay, with cells from progesterone (but not estradiol)-treated spayed rats. Such an in vitro system may provide a new opportunity to study the mechanism of decidualization and of its hormonal control.