Three types of transfection experiments were used to detect the abilities of different classes of antagonists to stimulate binding of progesterone receptor (PR) to progesterone response elements (PRE) in intact mammalian cells. These included a promoter interference assay, in which PR binding to PREs positioned between the TATA box and the start of transcription is detected as a reduction of expression of a constitutively active reporter gene, competition of PR antagonist and glucocorticoid receptor agonist for a common glucocorticoid response element/PRE-controlled reporter construct, and activation of a chimeric receptor (PR-VP16) containing the constitutive trans-activation domain derived from the VP16 protein of herpes simplex virus. By each approach, all antagonists tested were equally effective in stimulating PR binding to PREs in the cell. This included previously designated type I (ZK98299) and type II (RU486, ZK98734, and ZK112993) 11beta-aryl substituted steroid analogs. Stimulation of PR binding to PREs in the cell by ZK98299 was of interest because this antagonist has been reported to lack the ability to stimulate PR-DNA binding in vitro by electrophoretic gel mobility shift assay compared with RU486, which promotes efficient binding of PR to PREs. To clarify the apparent discrepancy between intact cell and in vitro results with ZK98299, we altered electrophoretic gel mobility shift assay conditions to allow detection of less stable DNA complexes. Under these conditions, ZK98299 induced the formation of specific PR-PRE complexes. Further analysis of the ZK98299-induced DNA complexes revealed that they exhibited an electrophoretic mobility different from that of the complexes induced by RU486, and the off-rate of PR from DNA was faster than that of the PR bound to agonist. This suggests that ZK98299 promotes a conformational change within PR distinct from that induced by RU486. The present results are consistent with the conclusions that ZK98299 stimulates PR binding to target DNA sequences and that ZK98299 and RU486 represent two mechanistic classes of antagonists based on inducing different conformational changes in PR.