Nitric oxide signaling is crucial for effecting long lasting changes in cells, including gene expression, cell cycle arrest, apoptosis, and differentiation. We have determined the temporal order of gene activation induced by NO in mammalian cells and have examined the signaling pathways that mediate the action of NO. Using microarrays to study the kinetics of gene activation by NO, we have determined that NO induces three distinct waves of gene activity. The first wave is induced within 30 min of exposure to NO and represents the primary gene targets of NO. It is followed by subsequent waves of gene activity that may reflect further cascades of NO-induced gene expression. We verified our results using quantitative real time PCR and further validated our conclusions about the effects of NO by using cytokines to induce endogenous NO production. We next applied pharmacological and genetic approaches to determine the signaling pathways that are used by NO to regulate gene expression. We used inhibitors of particular signaling pathways, as well as cells from animals with a deleted p53 gene, to define groups of genes that require phosphatidylinositol 3-kinase, protein kinase C, NF-kappaB, p53, or combinations thereof for activation by NO. Our results demonstrate that NO utilizes several independent signaling pathways to induce gene expression.