The rat chorioallantoic placenta is organized into two distinct zones (junctional and labyrinth) and expresses six members of the placental prolactin (PRL) family: placental lactogen-I (PL-I), PL-I variant (PL-Iv), PL-II, PRL-like protein-A (PLP-A), PLP-B, and PLP-C. These placental hormones are expressed in distinct cell- and temporal-specific patterns and can be used to monitor the state of differentiation of rat trophoblast cells. This study was initiated to examine the role of the fetus and maternal ovaries in the regulation of trophoblast cell differentiation and organization. Expression of the placental PRL family was monitored by Northern and Western blotting and immunocytochemical analysis. The roles of the fetus and maternal ovaries were examined by chemically induced fetal death (DFX, induced by intraamniotic injection of digoxin) and surgical removal of the ovaries (OVX), respectively. The endocrine differentiation of the placenta was assessed on day 19 of gestation (sperm positive = day 0, parturition = day 21). Day 10 of gestation was the earliest day that DFX could be reliably performed. Day 10 is a time point during pregnancy preceding the onset of expression of all members of the placental PRL family except PL-I. DFX on day 10 of gestation did not affect the endocrine differentiation of the trophoblast cells but did alter the organization of the chorioallantoic placenta. PL-II, PL-Iv, and the PLPs were all expressed in their appropriate molecular forms and cell types in placentas developing in the absence of fetal influence. The maternal ovaries, in the absence of the fetus, had two distinct actions on the placenta: ovarian signals were essential during a period at midgestation for the maintenance of the placenta and exposure of the developing placenta to ovarian signals during the second half of gestation, in the absence of the fetus, arrested labyrinth zone development. The active ovarian signals were progesterone and estrogen. We conclude that trophoblast cell differentiation occurs independent of the fetus and maternal ovaries. However, signals from both the fetus and maternal ovaries are required for normal organization of the chorioallantoic placenta.