The mouse blastocyst undergoes a program of protein secretion during the peri-implantation period including the initial production of placental lactogen-I (mPL-I) on day 6 of pregnancy. Although blastocysts collected on day 5 also produce mPL-I when cultured to form outgrowths on plastic dishes, it was not known if embryos have an intrinsic ability to produce mPL-I during culture in vitro, or if a specific uterine influence is necessary. Earlier experiments also suggested that attachment of the trophoblast to a stable surface may be a prerequisite for synthesis of mPL-I. Both questions were addressed by examining mPL-I production by day 3 and day 4 embryos cultured either on a two-dimensional tissue culture dish surface or in hanging drops. The presence of intracellular mPL-I was assayed by immunohistochemistry, while the secreted hormone was detected by its known position in two-dimensional electrophoresis gels. These experiments demonstrated that these earlier stage embryos do have an intrinsic program for mPL-I production which proceeds in vitro under various culture conditions. Synthesis of mPL-I occurred in embryos suspended in hanging drops as well as in those spreading on a solid two-dimensional surface, thus showing that adhesion to a surface is not required for production of this hormone. Although some mPL-I synthesis was seen in embryos cultured in medium containing BSA as the sole macromolecule, the inclusion of fibronectin either in dishes, where it supports attachment, or in the hanging drop system stimulated mPL-I secretion. Serum supplementation in both culture systems further increased growth and differentiation of the embryo, as well as mPL-I secretion, compared to fibronectin supplementation.