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-B, p53, or combinations thereof for activation by NO. Our results demonstrate that NO utilizes several independent signaling pathways to induce gene expression.Nitric oxide regulates a wide range of physiological responses (1). Most of the studies of NO action in animals have focused on its activity as an effector of rapid responses, such as regulation of blood pressure, smooth muscle contraction, and neurotransmission in the central and peripheral nervous systems. In these contexts, NO action is mediated by fast acting signaling cascades, which rapidly subside after the disappearance of the original stimulus (e.g. calcium influx).NO can also stimulate signaling pathways that elicit long lasting changes in the cell, and it can also reset genetic programs. This function of NO is manifested in its ability to regulate gene expression (2), to mediate immune response and inflammation (3), and to act as an antiproliferative factor to mediate tissue differentiation and organ development (4, 5). The molecular basis of these long lasting effects of NO is still unclear. To reveal the targets of NO in the cell, it will be important to understand the signaling pathways that are activated in response to NO.To investigate mechanisms underlying the long term action of NO, we used DNA microarrays to determine the temporal expression profiles of genes that respond to NO. We identified genes that are affected by NO and analyzed the temporal patterns of their expression to reveal cascades of NO-dependent gene regulation events. These results were verified by using quantitative real time PCR and validated by demonstrating that similar genes are activated in an NO-dependent manner in response to cytokines. We next used chemical inhibitors of specific signaling molecules and a mutant cell line to determine the signaling pathways that lead to the activation of spe...