The development of nitric oxide synthase (NOS) expression in the brain of Xenopus laevis tadpoles was studied by means of immunohistochemistry using specific antibodies against NOS and enzyme histochemistry for nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase. Both techniques yielded identical results and were equally suitable for demonstrating the nitrergic system in the brain. The only mismatches were observed in the olfactory nerve and glomeruli and in the terminal nerve; they were intensely labeled with the NADPH-diaphorase technique but failed to stain with NOS immunohistochemistry. As early as stage 33, nitrergic cells were observed in the caudal rhombencephalon within the developing inferior reticular nucleus. At later embryonic stages, different sets of reticular and tegmental neurons were labeled in the middle reticular nucleus and, more conspicuously, in the laterodorsal and pedunculopontine tegmental nuclei. As development proceeded, new nitrergic cell groups gradually appeared in the mesencephalon, diencephalon, and telencephalon. A general caudorostral temporal sequence was observed, both in the whole brain and within each main brain subdivision. The premetamorphic period was mainly characterized by the maturation of the cell populations developed in the embryonic period. During prometamorphosis, the nitrergic system reached an enormous development, and many new cell groups were observed for the first time, in particular in the telencephalon. By the climax of metamorphosis, the pattern of organization of nitrergic cells and fibers observed in the brain was similar to that present in the adult brain. Transient expression of NOS was not detected in any brain region. Our data suggest that nitric oxide plays an important role during brain development of Xenopus. Comparison with the developmental pattern of nitrergic systems in other vertebrates shows that amphibians possess more common features with amniotes than with anamniotes. J. Comp. Neurol. 445:59–77, 2002. © 2002 Wiley-Liss, Inc.