AbstractWe have used diaphorase histochemistry to study the morphology of cells expressing nitric oxide synthase in the superficial layers of the superior colliculus of developing and adult rats. The nitric oxide synthase‐positive cells showed a Golgi‐like morphology and were classified according to the cell types identified by several authors using the Golgi method. The first nitric oxide synthase‐positive cells appeared at postnatal day 7 and the number of stained cells increased progressively reaching a maximum at postnatal day 15. The poor staining of the dendritic tree and cell bodies in animals younger than postnatal day 15 allowed no unambiguous identification of the different cell types before that age. At postnatal day 15, based on cell soma and dendritic morphology, we have found that the following cell types express nitric oxide synthase: marginal, horizontal, narrow and wide‐field vertical and stellate. In the adult, the same cell types were found to express nitric oxide synthase but the staining intensity and frequency of each cell type was different from the developing animal. Our results show that cells expressing nitric oxide synthase constitute a subpopulation of neurons in which all cell types are represented. Furthermore, our observations of nitric oxide synthase expression by collicular cells starting by the end of the first postnatal week and reaching a maximum by postnatal day 15 parallels the functional development of the retino‐colliculur and cortico‐tectal projections and suggest that nitric oxide synthase‐positive cells might be involved in this process.