In the retina, nitric oxide (NO) functions in network coupling, light adaptation, neurotransmitter receptor function, and synaptic release. Neuronal nitric oxide synthase (nNOS) is present in the retina of every vertebrate species investigated. However, although nNOS can be found in every retinal cell type, little is known about the production of NO in specific cells or about the diffusion of NO within the retina. We used diaminofluorescein-2 (DAF-2) to image real-time NO production in turtle retina in response to stimulation with N-methyl-D-aspartate (NMDA). In response to NMDA, NO was produced in somata in the ganglion cell and inner nuclear layers, in synaptic boutons and processes in the inner plexiform layer, in processes in the outer plexiform layer, and in photoreceptor inner segments. This NO-dependent fluorescence production quickly reached transient peaks and declined more slowly toward baseline levels at different rates in different cells. In some cases, the NO signal was primarily confined to within 10 μm of the source, which suggests that NO may not diffuse freely through the retina. Such limited spread was not predicted and suggests that NO signal transduction may be more selective than suggested, and that NO may play significant intracellular roles in cells that produce it. Because NO-dependent fluorescence within amacrine cells can be confined to the soma, specific dendritic sites, or both with distinct kinetics, NO may function at specific synapses, modulate gene expression, or coordinate events throughout the cell.