The purpose of this investigation was to identify the cellular origin, and the temporal and regional characteristics of placental lactogen-I (PL-I) and placental lactogen-II (PL-II) expression during placental development in the rat. PL-I and PL-II mRNA expression were assessed by Northern blot analysis and in situ hybridization. PL-I and PL-II protein expression were determined by Western blot and immunocytochemical analyses. PL-I mRNA was first detected by in situ hybridization at Day 6 of gestation in mural trophoblast giant cells and a day later, PL-I protein was first detected by immunocytochemistry. PL-I immunostaining extended to the polar trophoblast giant cells as gestation advanced. Polar trophoblast giant cell staining for PL-I was not as intense as the mural trophoblast giant cell staining. Northern and Western blot analyses confirmed the asymmetric distribution of PL-I expression. PL-I mRNA migrated as a 1-kb species and PL-I protein migrated as 30- and 36-40-kDa forms. PL-I expression abruptly declined at Day 12, and by Day 13, PL-I was not detectable. PL-II protein was first detectable at Day 11 of gestation and was localized to trophoblast giant cells. PL-II mRNA could be detected at Day 10 of gestation. Northern and Western blot analyses indicated that PL-II expression significantly increased as gestation advanced and that PL-II expression was asymmetrically distributed similar to PL-I. PL-II mRNA migrated as a 1-kb species and PL-II protein migrated as a 25-kDa species. Blastocysts recovered on Day 4 of gestation initially showed no detectable expression of PL-I or PL-II; however, after 2 days of culture PL-I protein expression was detectable. Biochemical characteristics of PL-I synthesized and secreted by blastocyst outgrowths were similar to PL-I synthesized and secreted by Day 10 placental explants. In summary, (1) PL-I and PL-II are produced by trophoblast giant cells of the developing placenta, (2) PL-I and PL-II exhibit distinct temporal and regional patterns of expression during placental morphogenesis, and (3) PL-I expression by blastocyst outgrowths can be induced in vitro, whereas a more complex array of signals appears necessary for induction of PL-II expression.