Nitric oxide (NO) functions in biology as both a critical cytotoxic agent and an essential signaling molecule. The toxicity of the diatomic gas has long been accepted; however, it was not known to be a signaling molecule until it was identified as the endothelium-derived relaxing factor (EDRF). Since this discovery, the physiological signaling pathways that are regulated by NO have been the focus of numerous studies. Many of the cellular responses that NO modulates are mediated by the heme protein soluble guanylate cyclase (sGC). NO binds to sGC at a diffusion controlled rate, and leads to a several 100-fold increase in the synthesis of the second messenger cGMP from GTP. Other diatomic gases either do not bind (dioxygen), or do not significantly activate (carbon monoxide) sGC. This provides selectivity and efficiency for NO even in an aerobic environment, which is critical due to the high reactivity of NO. Several biochemical studies have focused on elucidating the mechanism of NO activation and O(2) discrimination. Significant advances in our understanding of these topics have occurred with the identification and characterization of the sGC-like homologues termed Heme-Nitric oxide and OXygen binding (H-NOX) proteins.