Cultured mouse blastocysts produce plasminogen activator, a protease that converts the zymogen plasminogen into the trypsin-like enzyme, plasmin. We have fractionated the blastocyst and cultured the constituent cell types. Trophoblast outgrowths free of inner cell mass derivatives secrete plasminogen activator during a time period that closely parallels the invasive phase of trophoblast cells in utero. Isolated inner cell masses also produce plasminogen activator; further fractionation of the inner cell mass as well as studies with primary cultures obtained from midgestation tissues demonstrate that enzyme formation is restricted entirely to parietal endoderm cells. Secretion of the enzyme may facilitate the migration of parietal endoderm cells along the trophoblast layer as the yolk sac cavity enlarges during gestation. F9 embryonal carcinoma cells do not secrete detectable amounts of plasminogen activator. However, when these cells are induced to differentiate, the resulting parietal endoderm-like cells are capable of producing the enzyme. These results are consistent with previous findings suggesting that plasminogen activator production may be a characteristic of invasive and/or migratory cells.