The mouse mammary tumor virus (MMTV) promoter has been used as a model to study how the glucocorticoid receptor (GR) remodels chromatin to allow other transcription factors to bind and activate transcription. To dissect the precise role of nuclear factor 1 (NF1) in chromatin remodeling and transcriptional activation, we used linker-scanning mutants of transcription factor binding sites on the MMTV promoter. We compared the NF1 mutant MMTV promoter in the context of transiently transfected templates (transient transfection) and templates organized as chromatin (stable transfection) to understand the effect of chromatin on factor binding and transcription. We show that on a transiently transfected template, mutation in the NF1 binding site reduces both basal and hormone-dependent transcription. This suggests that NF1 is required for transcription in the absence of organized chromatin. We also found that binding of NF1 on a transiently transfected template is independent of mutation in hormone response elements or the octamer transcription factor (OTF) binding site. In contrast, the binding of OTF proteins to a transiently transfected template was found to be dependent on the binding of NF1, which may imply that NF1 has a stabilizing effect on OTF binding. On a chromatin template, mutation in the NF1 binding site does not affect the positioning of nucleosomes on the promoter. We also show that in the absence of NF1 binding, GR-mediated chromatin remodeling of nucleosome B is reduced and hormone-dependent activation of transcription is abolished. Further, we demonstrate that NF1 is required for both the association of BRG1 chromatin remodeling complex and the GR on the promoter in vivo. These results suggest the novel possibility that NF1 may participate in chromatin remodeling activities in addition to directly enhancing transcription and that in the absence of its binding site the GR is unable to effectively bind the promoter and recruit the remodeling complex.