Nitric oxide (NO) is generated by a Ca2+/calmodulin-activated NO synthase and diffuses as a short-lived transcellular messenger through the plasma membrane. This study investigates the neurochemistry and anatomical distribution of NO-releasing cells in the CNS of the locust. Ca2+/calmodulin-activated NO synthase is responsible for fixation-sensitive NADPH diaphorase (NADPHd) activity in cell homogenates of the nervous system. Therefore, neurons expressing NO synthase were detected by NADPHd histochemistry performed in whole-mounts. The anatomical screening revealed fewer than 1% NADPHd-positive cells in the ventral nerve cord, some of which were single potentially identifiable neurons, and groups of cell bodies in several regions of the cerebral ganglion. A prominent feature of the histochemical survey in the cerebral ganglion is a group of 45 intensely stained cells innervating the olfactory neuropil of the antennal lobe. A basic requirement for identifying NO as a messenger molecule is the Ca(2+)-dependent release during nerve cell depolarization. With a sensitive photometric assay we demonstrated that dissociated cells from brain areas rich in NADPHd-positive neurons release NO after stimulation by agents elevating cytoplasmic Ca2+ levels and by the excitatory neurotransmitter acetylcholine. The combined anatomical and biochemical experiments therefore provide firm evidence that NO is a messenger molecule released in the CNS of the locust. Since locust neurons can be readily grown in primary culture, NO-induced elevations of CGMP levels and other signal transduction mechanisms in target cells will also be amenable to a cellular analysis.