Endometrial scrapings were collected from rat uteri at various times (0-4 days) after induction of a decidual reaction by i.p. injection of pyrathiazine hydrochloride (20 mg/animal) on the 5th day after a sterile mating. The tissue was dissociated by treatment with trypsin and the cells were cultured as monolayers. The differentiation of decidual cells was followed in these cultures. Two morphologically distinct cell populations were recognized: dispersed spindle-shaped or stellate cells and colonies of closely packed polygonal cells, 1st apparent after 48 h in culture. During culture, [3H]thymidine was incorporated into the nuclei of both cell types, as indicated by autoradiography. There was an increase in the number of cells in the colonies as culture progressed; most mitotic figures and the highest percentage of labeled nuclei were located within the colonies. Bi- and multinucleated cells, which are a characteristic feature of decidual tissue in vivo, appeared in the colonies on the 3rd day of culture and constituted about 60% of the colony population after 4-5 days. The dispersed cells showed a doubling in nuclear area during 4 days in culture, suggesting the formation of polyploid cells; such cells are prominent in fully differentiated decidual cells in vivo. The content of prostaglandin E in the cultured cell, determined by radioimmunoassay, was about 15-fold higher than that in rat embryo fibroblast cultures grown under similar conditions, and was comparable to the level of prostaglandins found in decidual cells in vivo.