The human granulocyte-macrophage colony-stimulating factor (GM-CSF) gene is activated by an NFAT-dependent enhancer forming an inducible DNase I hypersensitive (DH) site. The enhancer core comprising the DH site contains the GM330 and GM420 elements that bind NFAT and AP-1 cooperatively. Here we demonstrate that both elements are essential for enhancer activity and that Sp1 and AML1 sites in the enhancer become occupied in vivo only after activation. Chromatin structure analysis revealed that the GM-CSF enhancer core elements are divided between two adjacent nucleosomes that become destabilized and highly accessible after activation. Inducible chromatin reorganization was not restricted to the enhancer core but extended across a 3-kb domain of mobilized nucleosomes, within which the nucleosome repeat length was compressed from approximately 185 to 150 bp. The GM420 element is a high-affinity site that binds NFAT independently of AP-1 but depends on the linked AP-1 site for enhancer function. Nevertheless, just the NFAT motif from the GM420 element was sufficient to form a DH site within chromatin even in the absence of the AP-1 site. Hence, NFAT has the potential to cooperate with other transcription factors by promoting chromatin remodelling and increasing accessibility at inducible regulatory elements.