In vivo, transcription factors interact with promoters having complex nucleoprotein structures. The transiently expressed progesterone receptor (PR) efficiently activates a transfected mouse mammary tumor virus (MMTV) promoter but is a poor activator of the MMTV promoter when it acquires an ordered chromatin structure as an endogenous, replicating gene. We show that the deficiency in PR activity is not due to insufficient expression of either B or A isoforms or competition between the two types of MMTV templates. Rather, this deficiency reflects an inability to induce the chromatin remodeling event that is required for activation of the replicated MMTV template. To determine whether this characteristic is common to transiently expressed steroid receptors or specific to the PR, we examined the activity of transiently expressed glucocorticoid (GR) receptor. Unlike the PR, the transiently expressed GR is an effective activator of both MMTV templates and efficiently induces the necessary chromatin remodeling event at the replicated template. These results indicate that the GR and PR have unique requirements for activation of promoters with ordered chromatin structure. These differences may provide a mechanism for establishing target gene specificity in vivo for steroid receptors that recognize and bind to identical DNA sequences.