Nitric oxide is a gaseous, free radical molecule that functions in the nervous system as an atypical neurotransmitter, second messenger, vasodilator, and potent neurotoxin, depending on cell type and level of expression. Physiologically, nitric oxide is thought to mediate long-term potentiation, the cellular correlate of learning and memory formation, as well as synaptic plasticity and remodeling. Under pathologic conditions such as cerebral ischemia, NOS1 overproduces nitric oxide, which is a key mediator of excitotoxic cell death and neurodegeneration. Our laboratory discovered that multiple, individually functioning promoters (5’1 and 5’2) regulate human NOS1 gene expression. The present work describes the discovery and cloning of a novel human NOS1 promoter, 5’3, and the demonstration of its unique, developmentally regulated expression pattern in the central nervous system. Promoter-specific alternative splicing contributes to NOS1 mRNA diversity in both the 5’ untranslated and coding regions of the gene. We generated several lines of transgenic mice that express reporter genes under the control of separate human NOS1 promoter complexes (PR(5'1+5'2) and PR(5'3+5'4)), and studied how these alternate promoters contribute to NOS1 expression in various physiologic and pathophysiologic states in vivo. To this end, transgene expression is documented throughout normal development, which demonstrates overlapping but distinct patterns of promoter use that contribute to the overall recapitulation of endogenous NOS1 gene expression. In a murine model of striatal neurotoxicity, systemic methamphetamine administration results in promoter-specific transcriptional activation inthe transgenic mice. NOS1 enzymatic activity is stimulated in motor neuron cell bodies after peripheral transection of the facial nerve; however, the transgenic NOS1 promoter complexes are insufficient to recapitulate NOS1 activation in this model. These findings help clarify how multiple promoters and mRN A diversity contribute to the complex regulation of the human NOS1 gene, and further our general understanding of transcriptional regulation of gene expression via alternate promoters. Nitric oxide is a gaseous, free radical molecule that functions in the nervous system as an atypical neurotransmitter, second messenger, vasodilator, and potent neurotoxin, depending on cell type and level of expression. Physiologically, nitric oxide is thought to mediate long-term potentiation, the cellular correlate of learning and memory formation, as well as synaptic plasticity and remodeling. Under pathologic conditions such as cerebral ischemia, NOS1 overproduces nitric oxide, which is a key mediator of excitotoxic cell death and neurodegeneration. Our laboratory discovered that multiple, individually functioning promoters (5’1 and 5’2) regulate human NOS1 gene expression. The present work describes the discovery and cloning of a novel human NOS1 promoter, 5’3, and the demonstration of its unique, developmentally regulated expression pattern in the central nervous system. Promoter-specific alternative splicing contributes to NOS1 mRNA diversity in both the 5’ untranslated and coding regions of the gene. We generated several lines of transgenic mice that express reporter genes under the control of separate human NOS1 promoter complexes (PR(5'1+5'2) and PR(5'3+5'4)), and studied how these alternate promoters contribute to NOS1 expression in various physiologic and pathophysiologic states in vivo. To this end, transgene expression is documented throughout normal development, which demonstrates overlapping but distinct patterns of promoter use that contribute to the overall recapitulation of endogenous NOS1 gene expression. In a murine model of striatal neurotoxicity, systemic methamphetamine administration results in promoter-specific transcriptional activation inthe transgenic mice. NOS1 enzymatic activity is stimulated in motor neuron cell bodies after peripheral transection of the facial nerve; however, the transgenic NOS1 promoter complexes are insufficient to recapitulate NOS1 activation in this model. These findings help clarify how multiple promoters and mRN A diversity contribute to the complex regulation of the human NOS1 gene, and further our general understanding of transcriptional regulation of gene expression via alternate promoters.