It has been suggested that ligand-dependent gene activation by the progesterone receptor (PR) can result from recruitment of PR by the promoter bound Sp1. A detailed investigation of the Sp1-dependent agonistic activity of RU486 and R5020 on the folate receptor (FR) type , p27, thymidine kinase 1 (TK1) and p21 genes reveals a different mechanism. The FR- P4 promoter and the endogenous FR- gene were up-regulated by the PR agonist R5020 through either PR-A or PR-B. The classical antagonist RU486 also activated the promoter but only through PR-B. The most proximal (essential) G/C-rich (Sp1 binding) element and the initiator region constituted the minimal promoter responsive to PR regulation; substitution with a stronger cluster of G/C-rich elements enhanced the magnitude of the PR response. In contrast, substitution of the G/C-rich element with a TATA box resulted in the loss of regulation by PR. Over-expression of Sp1 and Sp4 but not Sp3 enhanced activation of the FR- promoter by PR; knocking down Sp1 decreased the activation in a manner that was reversed by ectopic Sp1 or Sp4. The ligand-dependent action of PR on the promoter was delayed compared to its activation of a classical GRE-driven promoter and activation of both the promoter and the endogenous FR- gene by PR required new protein synthesis. Activation by PR paralleled RNA polymerase II recruitment but was not accompanied by either association of PR or a change in the association of Sp1 with the endogenous FR- P4 promoter. Similar observations were made for PR regulation of the genes encoding p27, TK1 and p21. The results contradict the current view of Sp1-dependent gene regulation by PR and point to the existence of a PR target gene(s) whose promoter and cell context(s) must thus be key determinants of the agonistic activity of RU486 on a large group of important Sp1-dependent downstream target genes.