Higher order mental processes, such as learning, memory, and cognition, are built upon the brain's remarkable capacity to convert everyday, transient experiences into stored memories that can last a lifetime. Specific biochemical and structural changes at synapses, the highly specialized points of communication between neurons, are thought to serve as the molecular basis for these processes. However, the underlying mechanisms that regulate these synaptic modifications are poorly understood. For example, it remains unclear how synaptic activity regulates specific patterns of gene expression required to maintain these long-term adaptive synaptic changes. To gain insight into these mechanisms, this study investigated the transcriptional regulation of Arc, a neuronal immediate-early gene (IEG) essential for synaptic plasticity. Several powerful techniques, which had not previously been used in combination to study the transcription of a neuronal IEG, were used here to identify and characterize the genomic regulatory regions directly involved in Arc transcription. The DNaseI hypersensitivity assay was first applied in primary rat cortical neuron cultures to screen the endogenous chromatin structure flanking the Arc gene locus for discrete "open" nucleosome-depleted regions. A novel reporter gene driven by these putative regulatory regions was then used to identify two transcriptional enhancer regions located ~6.5 kilobase pairs (Kb) and ~850-950 base pairs (bp) upstream of Arc. Bioinformatic analysis was then used to help identify an evolutionarily and functionally conserved serum response element (SRE) within the distal enhancer, as well as a conserved, yet non-canonical binding site for a putative novel neuronal transcription factor within the proximal enhancer. Biochemical binding studies confirmed that the serum response factor binds to this SRE in vivo at the endogenous Arc promoter, and that some unknown nuclear protein(s) binds in vitro to the proximal enhancer region. Finally, two additional regulatory regions, located ~9.3 Kb and ~700-850 bp upstream of Arc, with apparent repressor activity were also identified. Thus, this investigation provides novel insights into the molecular mechanisms underlying the regulation of Arc gene expression.