The distribution of the neuronal type of nitric oxide synthase in the goldfish brain and spinal cord was investigated via NADPH-diaphorase histochemistry and immunocytochemistry using an antiserum raised against the purified mammalian enzyme. Many structures, including magnocellular neurosecretory cells, motoneurons, mesencephalic trigeminal neurons, and radial glial fibers, were stained by the NADPH-diaphorase reaction but were not immunoreactive. This nonspecific NADPH-diaphorase activity was strongly reduced after preincubation of the sections. Therefore, when sections were first reacted for immunofluorescence and, thereafter, stained for NADPH-diaphorase, a corresponding staining pattern was obtained that allowed the reliable localization of neuronal nitric oxide synthase based on both complementary staining methods. In the telencephalon, positive neurons were concentrated in the ventral and posterior parts of the area ventralis. Many intensely stained neurons were present in various diencephalic nuclei, including the nucleus centralis posterior and the ventromedial nucleus of the thalamus, the nucleus tori lateralis, the nucleus recessus lateralis, the nucleus tuberis posterior, and the central nucleus of the inferior lobe. In the midbrain, neurons containing nitric oxide synthase were located in the periventricular zone of the optic tectum, the nucleus vermiformis, and the nucleus reticularis mesencephali. Specific staining in the cerebellum was concentrated in Golgi cells. In the hindbrain, nitroxergic neurons were numerous in all four sensory nuclei of the trigeminus, in the facial lobe, the superior olive, the inferior reticular formation, and the medial general visceral nucleus of the vagus. The dorsal horn of the spinal cord was enriched with positive neurons. A few strongly stained cells were also present in the ventral horn. In conclusion, neurons capable of synthesizing nitric oxide occur throughout the teleost central nervous system. The presence of nitric oxide synthase in projection areas of most afferent nerves suggests a widespread involvement of nitric oxide in sensory information processing. The distribution of nitric oxide synthase-containing neurons in certain areas, e.g., the tectum opticum and the spinal cord, indicates an evolutionarily conserved pattern. Similar to the case in other vertebrates, there appears to be no comprehensive overlap between the distribution of nitric oxide synthase and that of any other chemically characterized neuronal population described thus far. However, strongly positive cell groups in the mesencephalic reticular formation suggest the idea of an evolutionarily conserved mesopontine cholinergic system coexpressing nitric oxide synthase.