Expressional dysregulation of the human neuronal nitric oxide synthase (NOS1) gene represents an important mechanism in the pathogenesis of certain neuronal disease states. The structure and regulation of the human NOS1 gene is highly complex based on cell type- and stimulus-dependent usage of multiple exon 1 variants. Here we demonstrate that the untranslated region of exon 2 exerts promoter and enhancer functions as well, facilitated in large part by cooperative interaction of two conserved adjacent CREB/AP-1 binding sites. In human neuronal A673 cells, NOS1 expression is stimulated by several compounds which act through these sites, but also stimulate the combined promoter region of exons 1f and 1g. While stimulation of NOS1 expression by dibutyryl-cAMP is mediated by protein kinase A (blocked by H-89), the antiepileptic drug valproic acid is likely to activate phosphatidylinositol-3 kinase (inhibited by LY 294002).