Nitric oxide (NO) is a versatile signaling molecule with diverse biological activities, influenced by its concentration and chemical reactions. The authors propose five distinct concentration levels of NO activity: cGMP-mediated processes (<1–30 nM), Akt phosphorylation (30–100 nM), stabilization of HIF-1α (100–300 nM), phosphorylation of p53 (>400 nM), and nitrosative stress (1 μM). Lower NO concentrations promote cell survival and proliferation, while higher levels favor cell cycle arrest, apoptosis, and senescence. Free radical interactions, particularly reactive oxygen species (ROS), can reduce NO concentrations, antagonizing its signaling and sometimes converting cell cycle arrest to survival. The chemical biology of NO is divided into direct and indirect reactions, with direct reactions occurring at low concentrations and indirect reactions at higher concentrations. Indirect effects can be further categorized into nitrosative and oxidative stress, with the balance between these processes determined by NO concentration. The concentration dependence of NO responses is crucial, with low concentrations promoting pro-growth and anti-apoptotic responses, while higher concentrations favor cytostatic and apoptotic pathways. Temporal aspects of NO exposure, such as immediate and delayed responses, also play a role in signaling. The concentration range of endogenously generated NO can vary widely, from sub-nM to μM levels, depending on the biological context and the source of NO production. Factors influencing NO concentration include production rates, diffusion distance, consumption rates, and reactivity with target molecules. These kinetic determinants shape the complex and context-dependent responses of cells to NO.Nitric oxide (NO) is a versatile signaling molecule with diverse biological activities, influenced by its concentration and chemical reactions. The authors propose five distinct concentration levels of NO activity: cGMP-mediated processes (<1–30 nM), Akt phosphorylation (30–100 nM), stabilization of HIF-1α (100–300 nM), phosphorylation of p53 (>400 nM), and nitrosative stress (1 μM). Lower NO concentrations promote cell survival and proliferation, while higher levels favor cell cycle arrest, apoptosis, and senescence. Free radical interactions, particularly reactive oxygen species (ROS), can reduce NO concentrations, antagonizing its signaling and sometimes converting cell cycle arrest to survival. The chemical biology of NO is divided into direct and indirect reactions, with direct reactions occurring at low concentrations and indirect reactions at higher concentrations. Indirect effects can be further categorized into nitrosative and oxidative stress, with the balance between these processes determined by NO concentration. The concentration dependence of NO responses is crucial, with low concentrations promoting pro-growth and anti-apoptotic responses, while higher concentrations favor cytostatic and apoptotic pathways. Temporal aspects of NO exposure, such as immediate and delayed responses, also play a role in signaling. The concentration range of endogenously generated NO can vary widely, from sub-nM to μM levels, depending on the biological context and the source of NO production. Factors influencing NO concentration include production rates, diffusion distance, consumption rates, and reactivity with target molecules. These kinetic determinants shape the complex and context-dependent responses of cells to NO.