Nitric oxide (^•NO) is a labile endogenous free radical produced upon glutamatergic neuronal activity in hippocampus by neuronal nitric oxide synthase (nNOS), where it acts as a modulator of both synaptic plasticity and cell death associated with neurodegeneration. The low CNS levels and fast time dynamics of this molecule require the use of rapid analytical methods that can more accurately describe its signaling in vivo. This is critical for understanding how the kinetics of ^•NO‐dependent signaling pathways is translated into physiological or pathological functions. In these studies, we used ^•NO selective microelectrodes coupled with rapid electrochemical recording techniques to characterize for the first time the concentration dynamics of ^•NO endogenously produced in hippocampus in vivo following activation of ionotropic glutamate receptors. Both L‐glutamate (1–100 mM) and N‐methyl‐D‐aspartate (NMDA; 0.01–5 mM) produced transient, dose‐dependent increases in extracellular ^•NO concentration. The production of ^•NO in the hippocampus by glutamate was decreased by the nNOS inhibitor 7‐NI. Intraperitoneal administration of the NMDA receptor blocker, MK‐801, and the inhibitor of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoazolepropionic acid (AMPA) receptor, NBQX, applied locally greatly attenuated glutamate‐evoked overflow of ^•NO. Thus, ^•NO overflow elicited by activation of glutamate receptors appeared to result from an integrated activation of ionotropic glutamate receptors, both of the NMDA and AMPA receptors subtypes. Additionally, distinct concentration dynamics was observed in the trisynaptic loop with stronger and longer lasting effects of glutamate activation on ^•NO overflow seen in the CA1 region as compared with the dentate gyrus. Overall, the results provide a quantitative and temporal basis for a better understanding of ^•NO activity in the rat hippocampus. © 2010 Wiley‐Liss, Inc.