Mouse placental lactogen-I (mPL-I) is the earliest appearing member of the PRL-GH family of placental hormones. Using immunological techniques, we have localized mPL-I both in utero andin vitro to trophoblast giant cells. Detectable amounts of mPL-I were first seen by immunohistochemistry on day 6 of gestation in the mural trophoblast. On day 7 giant cells of the ectoplacental cone also contained mPL-I, and on day 10 giant cells adjacent to both the decidua basalis and decidua capsularis stained positive. By day 12 however, all intra- and extraembryonic tissues were negative. The pattern of mPL-I staining in the placenta is consistent with its reported gestational serum profile. mPL-I was also present in trophoblast giant cells in vitro shortly after attachment and spreading of day 5 blastocysts. Analysis of conditioned medium from blastocyst outgrowths by immunoprecipitation and two-dimensional electrophoresis, and by immunoblotting, identified mPL-I as the major secretory product from the trophoblast, consisting of a group of acidic proteins of 30,000–45,000 Mr. The synthesis and secretion of mPL-I in vitro occurred in a serum-free medium suggesting that production of this hormone is part of an endogenous program of trophoblast differentiation. (Endocrinology 126: 2809–2814, 1990)