Recent work in nonmammalian vertebrate retinas has suggested that other cell types besides the generally accepted amacrine cells may contain serotonin. We have used immunocytochemical methods to study serotonin-like immunoreactivity (5-HTLI) in the retina of the developing and mature frog Rana pipiens. In the adult, two types of serotonin immunoreactive (5-HT-ir) cells were found in the inner nuclear layer (INL) of the retina. Additionally, a large population of cells in the retinal ganglion cell layer (RGCL) had 5-HTLI. These cells were grouped into three types based on their soma size and their primary dendritic branching pattern. The optic nerve fiber layer was also intensely stained with serotonin antisera although staining intensity decreased progressively as the fibers approached the optic nerve head. Severing the optic nerve resulted in 5-HT-ir elements that extended up the optic nerve shaft from the lesion site toward the retina. Both regional and temporal changes in the pattern of 5-HTLI were seen. In middle regions of retina, approximately 30% of the cells in the RGCL were 5-HT-ir. Nasal and temporal regions of central retina had significantly fewer 5-HT-ir cells. Early in development only scattered cells in the RGCL were 5-HT-ir. As the animals matured there was an increase in both the proportion and the staining intensity of these cells. Our results suggest that in studying the function and development of the visual system in this animal, the role of serotonin must be examined.