The effect of steroid hormones on multiplication of the human polyomavirus BK (BKV) was studied. Physiological concentrations of the synthetic glucocorticoid dexamethasone, progesterone R5020, or estrogen 17 beta-estradiol enhanced the permissivity of the host cell for BKV, resulting in an up to 11-fold (dexamethasone), 5-fold (progesterone), or 3-fold (17 beta-estradiol) higher virus yield. The increase in virus yield in dexamethasone-stimulated cells correlated with enhanced steady-state levels of viral transcripts. The late leader sequence of the BKV control region contains a hormone response unit composed of a nonconsensus glucocorticoid and/or progesterone response element (GRE/PRE) and a fully consensus estrogen response element (ERE). DNA-protein binding studies showed that the glucocorticoid receptor and the progesterone receptor bound to this BKV GRE/PRE-like sequence, while the estrogen receptor could bind to the BKV ERE motif. By transient transfection assays, we were able to show that these sequences can mediate steroid hormone-induced gene expression. However, no cooperative transactivation effect between the BKV GRE/PRE-like motif and BKV ERE motif was observed. This BKV hormone response unit may play an important role in vivo by enhancing a productive BKV infection, and perhaps also by reactivating a latent infection, during physiological or pathological conditions accompanied by increased steroid hormone levels.