A 2.4-kilobase (kb) DNA fragment, located 7.1 kb upstream from the human tissue-type plasminogen activator (t-PA) gene (t-PA2.4), acts as an enhancer which is activated by glucocorticoids, progesterone, androgens, and mineralocorticoids. Transient expression of t-PA-chloramphenicol acetyltransferase reporter constructs in HT1080 human fibrosarcoma cells identified a glucocorticoid responsive unit with four functional binding sites for the glucocorticoid receptor, located between bp -7,501 and -7,974. The region from bp -7,145 to -9,578 (t-PA2.4) was found to confer a cooperative induction by dexamethasone and all-trans-retinoic acid (RA) to its homologous and a heterologous promoter, irrespective of its orientation. The minimal enhancer, defined by progressive deletion analysis, comprised the region from -7.1 to -8.0 kb (t-PA0.9) and encompassed the glucocorticoid responsive unit and the previously identified RA-responsive element located at -7.3 kb (Bulens, F., Ibañez-Tallon, I., Van Acker, P., De Vriese, A., Nelles, L., Belayew, A., and Collen, D. (1995) J. Biol. Chem. 270, 7167-7175). The amplitude of the synergistic response to dexamethasone and RA increased by reducing the distance between the enhancer and the proximal t-PA promoter. The synergistic interaction was also observed between the aldosterone and the RA receptors. It is postulated that the t-PA0.9 enhancer might play a role in the hormonal regulation of the expression of human t-PA in vivo.