Sertoli cells secrete plasminogen activators (PAs) on both sides of the blood-testis barrier, i.e., in the basal and apical compartments of the seminiferous tubules, whereas peritubular cells secrete plasminogen activator inhibitor-1 (PAI-1), a fast-acting and specific PA inhibitor. While it is likely that PAI-1 produced by peritubular cells counteracts the basal secretion of PA, the nature of the PA inhibitor acting in the apical compartment remains to be demonstrated. In the present study, we showed that Sertoli cells recovered from 20-day-old rats and cultured contained a transcript of 3-3.2 kilobases, which hybridized specifically to a PAI-1 cDNA probe (Northern blot). We verified that the observed PAI-1 transcript could not result solely from the peritubular cells (weakly contaminating the Sertoli cell cultures), by comparing PAI-1 mRNA levels of Sertoli and peritubular cells recovered from 20-day-old rats and cultured. We also demonstrated that cultured Sertoli cells secreted a protein that complexed with tissue-type PA (zymography), indicating that it was biologically active. This protein comigrated with purified PAI-1 as a doublet of 46 and 49 kDa (Western blot). The trophic hormone FSH decreased PAI-1 messenger RNA as well as immunoreactive PAI-1 protein (probably via the cAMP protein kinase A pathway), whereas transforming growth factor ss1 and basic fibroblast growth factor (in a nanomolar concentration) increased both of these. These observations support the hypothesis that PAI-1 is expressed by Sertoli cells and is under a complex hormonal (FSH) and paracrine and/or autocrine control exerted at least by basic fibroblast growth factor and transforming growth factor ss1.