Tissue-type plasminogen activator (tPA) activates plasminogen to the active protease plasmin and is implicated in many biological processes that require extracellular proteolysis. In rat ovarian cells, gonadotropins induce the tPA gene by a cAMP-dependent pathway and this induction correlates with the time of follicular rupture. We have previously identified several promoter elements within the first 621 bp of the rat tPA promoter that are important for constitutive and cAMP-induced expression of the gene, including a cAMP responsive element (CRE), a nuclear factor 1 (NF1) element, a SP1-binding site and a G+C-rich box. In this report we have extended our study by analysing promoter constructs, ranging in size from 7.7 kb to 135 bp fused to the luciferase reporter gene. Transient transfection analysis of rat granulosa cells and human 293 cells, reveal that the proximal 268 bp of the promoter is enough to confer high basal and cAMP-induced expression of the gene. At position -162 to -172, between the previously identified CRE and NF1 sites, a novel TAAT-containing promoter element was identified. Mutational inactivation of the TAAT motif indicates that this element is important for both constitutive and cAMP-induced expression of the gene, and for the binding of a presumably novel nuclear factor that we have termed tPA promoter factor-1 (tPF-1).