Nitric oxide (NO), a cellular signaling molecule, is produced in the brain by both neurons and astrocytes. While neurons are capable of rapid release of small amounts of NO serving as neurotransmitter, astrocytic NO production has been demonstrated mainly as a slow reaction to various stress stimuli. Little is known about the role of astrocyte-produced NO. Using the NO indicator 4,5-diaminofluorescein-2 diacetate (DAF-2DA) and acute slices from mouse brain, we distinguished neurons from astrocytes based on their different fluorescence kinetics and pattern, cellular morphology, electrophysiology, and responses to selective nitric oxide synthase (NOS) inhibitors. Typically, astrocytic fluorescence followed neuronal fluorescence with a delay of 1–2 min and was dependent on the inducible NOS isoform (iNOS) activity. Western blot analysis established the presence of functional iNOS in the neocortex. An assay for cell death revealed that most DAF-2DA-positive neurons, but not astrocytes, were damaged. Whole cell recordings from astrocytes confirmed that these cells maintained their membrane potential and passive properties during illumination and afterward. Induction of excitotoxicity by brief application of glutamate triggered an immediate and intense astrocytic response, while high-frequency electrical stimulation failed to do so. The present study demonstrates, for the first time, rapid and massive iNOS-dependent NO production by astrocytes in situ, which appears to be triggered by acute neuronal death. These data may bear important implications for our theoretical understanding and practical management of acute brain insults. © 2005 Wiley-Liss, Inc.