The distribution of nitric oxide synthase (NOS) in the lamprey brain was studied by using reduced nicotinamide-adenine-dinucleotide phosphate (NADPH)-diaphorase histochemistry to further elucidate the evolution of neurons synthesizing nitric oxide. Intense labeling of fibers and/or neurons was found in portions of the lamprey central nervous system, such as the olfactory system, the pineal organ, the habenular region, the nervus stato-acousticus (N. VIII), the brainstem, and the spinal cord, and also in the adenohypophysis. Labeled giant cells located at the floor of the 3rd and 4th ventricle were recognized as reticulospinal neurons. Mauthner and Müller cells were identified according to morphological criteria. Eight pairs of Müller cells and one pair of Mauthner cells were labeled by NADPH histochemistry. None of these cells had, as yet, been described to display NOS activity in any vertebrate. The massive staining of these cells and the apparent lack of labeling, e.g., in teleost fishes, may be a histochemical correlate to already known differences of functions served by these cells in different species. In addition, our results suggest that the nitric oxide (NO) system has appeared early in vertebrate evolution.