The hormonal regulation of the human urokinase type plasminogen activator (uPA) gene has been studied by introducing into mouse and rat Sertoli cell primary cultures a recombinant plasmid, in which the transcription regulatory elements of the cloned human uPA gene drive the expression of the bacterial chloramphenicol-acetyl-transferase gene. It was found to be expressed and regulated by FSH and (Bu)2cAMP in the mouse cells only, in agreement with data on the expression of the endogenous gene in rat and mouse gonads. The stimulation of transcription by FSH was evident in cultures from 13-day-old but not from 18-day-old mice, even though (Bu)2cAMP induction could be observed at both ages. Phorbol-myristate acetate was found to activate the human uPA promoter in Sertoli cell cultures from mice of both ages, even though the effect was less evident in cultures of 18-day-old animals. Deletion analysis of the human uPA 5'-flanking region showed that the distal enhancer element is not needed for (Bu)2cAMP induction, and that at least two promoter regions are involved in (Bu)2cAMP induced transcription. One of these cAMP responsive regions lies between nucleotides -72 and -29 from the CAP site. The sequence of this region would suggest the binding of transcription factor AP-2, a cell-specific mediator of both cAMP and phorbol esters action on gene expression. However, these sequences do not mediate phorbol ester activation of human uPA promoter in mouse Sertoli cells.