Variations in the production of plasminogen activator (PA), a proteolytic enzyme that has been associated with trophoblast invasiveness, may be critical to the success or failure of placentation. To understand better the mechanisms regulating PA production we have cultured human trophoblast cells and measured the effect of steroid hormones and protein synthesis inhibitors upon their capacity to lyse 125I-fibrinogen. Progesterone (P) and 17 beta-estradiol (E2) at concentrations of 10(-8)M, 10(-7)M, and 10(-5)M did not cause significant changes in PA or chorionic gonadotropin (hCG) production by the trophoblast cells. In contrast, dexamethasone (10(-8)M and 10(-5)M) caused a significant, dose-dependent decrease in PA production. There was no significant difference in PA and hCG production between trophoblast cultures treated with cytosine arabinoside (1 mg/ml) and untreated cultures. When actinomycin D (0.5 microgram/ml) was added to cultures at time zero, the production of PA and hCG during the first 24 hours of incubation was not significantly different from that of untreated cultures. After 24 hours, however, PA and hCG disappeared from the actinomycin-treated cultures while both substances continued to be released in the untreated controls. These experiments indicate that trophoblastic PA and hCG production in vitro are independent of DNA replication and not under P or E2 control. They also show that PA and hCG are stored within the trophoblast cells and released into the medium during the first 24 hours of incubation; after this period the presence of PA and hCG in the medium represents de novo synthesis. The data also suggest that, similar to hCG, PA is produced continuously in vitro as long as the trophoblast cell is functionally active.