We characterized regulation of the human monocyte chemoattractant protein-1 (hMCP-1) gene by IFN-gamma in astrocytoma cells, because astroglial cells express chemokines in several central nervous system inflammatory states. It was found that IFN-gamma-induced hMCP-1 transcription was rapid, transient, and mediated by a 213-bp promoter-proximal regulatory region of the gene. Our studies on both in vitro and in vivo states of the hMCP-1 regulatory region established requirement of an IFN-gamma-activated site (GAS) and the presence of IFN-gamma-inducible GAS-binding activity involving at least STAT-1alpha for IFN-gamma-induced hMCP-1 expression. Unexpectedly, in vivo genomic footprinting of the proximal regulatory region of the IFN-gamma-induced gene revealed protection of a GC-rich sequence (GC box) with the same temporal pattern as that seen at the GAS; in vitro, this GC-rich element is associated with nuclear factor Sp1. These observations suggested a cooperative interaction between the GAS and the GC box element. Interestingly, site-specific mutations that abolished GC-box or GAS-element function produced clearly disparate results. Disruption of the GC box did not affect fold induction by IFN-gamma but reduced promoter-reporter expression by half. Conversely, GAS mutation abrogated induction but did not affect the magnitude of expression. These results establish the importance of the GAS element for induction of hMCP-1 and further our understanding of IFN-gamma-mediated transcriptional induction by providing the first evidence in vivo for inducible signaling to the GC box by this cytokine.