Accessing binding sites in DNA wrapped around histones in condensed chromatin is an obstacle that transcription factors must overcome to regulate gene expression. Here we demonstrate cooperativity between two transcription factors, the glucocorticoid receptor (GR) and nuclear factor 1 (NF1) to bind the mouse mammary tumor virus promoter organized as regular chromatin in vivo. This cooperativity is not observed when the promoter is introduced transiently into cells. Using RNA interference to deplete NF1 protein levels in the cells, we confirmed that NF1 promotes binding of GR to the promoter. Furthermore, we observed a similar synergism between GR and NF1 binding on the endogenous 11beta-hydroxysteroid dehydrogenase promoter, also regulated by GR and NF1. Our results suggest that the chromatin architecture of the promoters does not permit strong association of GR in the absence of NF1. Therefore we propose that cooperativity among DNA binding factors in binding to their cognate recognition sites in chromatin may be an important feature in the regulation of gene expression.