The trophoblast giant cell lineage is characterized by endoreduplication and expression of members of the PRL gene family. This report describes the functional consequences following in vitro manipulation of a rat trophoblast cell line, termed Rcho-1. Rcho-1 cells can be cultured under conditions that promote proliferation or differentiation. Proliferation is maintained by culturing the cells in the presence of fetal bovine serum under subconfluent conditions. Differentiation is induced by growing the cells to confluence and removing the mitogenic source. Differentiation is characterized by continued synthesis of DNA in the absence of proliferation (endoreduplication) and the sequential expression of members of the PRL gene family. Western and Northern blot analyses demonstrated that placental lactogen-I (PL-I) was first expressed, followed sequentially by PL-II, PRL-like protein-A, and PRL-like protein-C. The ontogeny of expression of members of the PRL gene family by the Rcho-1 cells recapitulated the pattern of in situ expression by trophoblast giant cells of the junctional zone of the chorioallantoic placenta. A notable difference between in vivo trophoblast giant cell differentiation and in vitro Rcho-1 cell differentiation is the termination of PL-I expression in normal trophoblast giant cells developing in vivo and the continued expression of PL-I in differentiated Rcho-1 cell cultures. The Rcho-1 cell line provides a unique in vitro model for investigating the initiation and maintenance of the trophoblast giant cell differentiation pathway.