The mechanisms underlying nitric oxide ((•)NO) synthesis and inactivation in the brain are essential determinants of (•)NO neuroactivity. Although (•)NO production is well characterized, the pathways of inactivation in vivo remain largely unknown. Here, we characterize the kinetics and the major mechanism of (•)NO inactivation in the rat brain cortex and hippocampus in vivo by measuring locally applied (•)NO with carbon-fiber microelectrodes (CFMs) and ceramic-based microelectrode arrays (MEAs). An apparent first-order clearance was observed in both brain regions, with decay rate constants (k) of (•)NO signals of 0.67 to 0.84 per second, significantly higher than the k obtained in agarose gel (0.099 per second), used as a (•)NO diffusion-control medium. (•)NO half-life in vivo, estimated by mathematical modeling, was 0.42 to 0.75 s. Experiments using MEAs support that the (•)NO diffusion radius is heterogeneous and related to local metabolic activity and vascular density. After global ischemia, k decreased to control values of diffusion in gel, but during anoxia, k decreased only 21%. Additionally, k in brain slices was threefold to fivefold lower than that in vivo, and hemorrhagic shock induced a 53% decrease in k. Overall, the results support that (•)NO scavenging by circulating erythrocytes constitutes the major (•)NO-inactivation pathway in the brain.