Tissue type (t) and urokinase type (u) plasminogen activators (PA) have been shown to be secreted by Sertoli cells in the seminiferous tubules in a cyclic fashion and to be dependent upon hormonal stimulation or the presence of adjacent spermatogenic cells. Furthermore, in cultured Sertoli cells, tPA has been shown to respond to FSH induction whereas uPA appears to be nonresponsive to gonadotropins. In the present study we analyzed the production of PA by Sertoli cells and regulation of this production by FSH during puberty. Cultured Sertoli cells under basal conditions secreted predominantly uPA. This production was high in 10-day-old animals and gradually decreased in older animals. The treatment of cultures with FSH or dibutyl cAMP induced production of tPA by Sertoli cells but at the same time produced a decrease in uPA activity. Northern blot analysis revealed that the control of PA synthesis is at the steady-state level of their mRNAs. Moreover, the use of cycloheximide, a protein synthesis inhibitor, showed that while tPA stimulation did not require intermediary protein synthesis, the decrease in uPA production was dependent upon protein synthesis. The differences in PA production by Sertoli cells obtained after FSH stimulation could explain the cyclic production of the two enzymes during the spermatogenic cycle and reinforce the hypothesis that different roles are played by the two enzymes in the several events occurring in testis development.