During early pseudopregnancy, DNA synthesis and mitosis in the uterine endometrial stroma precede the development of uterine sensitivity to deciduogenic stimuli. Progesterone redirects the effects of estradiol on endometrial DNA synthesis from the luminal epithelium to the stroma. To determine the time and hormonal control of preimplantation endometrial DNA synthesis, uterine cells were labeled with [3H] thymidine at specific times during early pseudopregnancy or after progestin and estrogen treatment of ovariectomized rats. The fate of these labeled cells after their decidualization was examined by separation of prelabeled deciduomal cells by velocity sedimentation at unit gravity, which separates cells by size. Stromal cells that synthesized DNA during early pseudopregnancy or in response to hormone treatment later differentiated into deciduomal cells. Rates of DNA synthesis increased on days 4 and 5 of pseudopregnancy, with greater incorporation occurring on day 4 in cells that differentiated into polyploid deciduomal cells. In ovariectomized rats, medroxyprogresterone acetate treatment for 15 h increased DNA synthesis in stromal cells that differentiated into diploid and tetraploid deciduomal cells. DNA synthesis increased further at 30 h before returning to basal levels at 48 h. After progestin pretreatment, estradiol treatment increased stromal DNA synthesis again with greater incorporation in cells differentiating into polyploid deciduomal cells. These data indicate that during early pseudopregnancy, both progesterone and estradiol control the DNA synthesis of endometrial stromal cells as a means of reprogramming these cells for the later growth and differentiation of decidualization.