The reticular formation contributes serotonin to many brain regions, including the optic tectum. We examined the organization and development of its serotonergic neurons in the leopard frog. Serotonin-immunoreactive (5-HT-ir) cells in adult frogs were organized into 10 distinct populations that were identified on the basis of their location and cellular morphology. Populations ranged in size from 16 to 2,066 cells and sometimes spanned more than one previously identified nuclear region. Four of the ten populations were absent in tadpoles. The remaining populations, though present, had two contrasting patterns of development. Half of the populations were established early and showed little change in numbers during tadpole stages but increased in size in juvenile and adult frogs. The other half increased dramatically during tadpole stages but failed to add many more cells in juveniles and adults. Three populations provided 90% of the serotonergic projections from the reticular region to the adult optic tectum. These projections were established early in development and likely originated from the dorsal raphe, median raphe, raphe pontis, raphe magnus, and reticularis pontis oralis. Termination sites were located in midtectal layers and were not topographically organized. We conclude that serotonergic cells within the reticular formation of the leopard frog have an organization similar to that found in mammals, that the overall increase in numbers of these cells is attributable to growth in different cell populations at different stages, and that input from this region changes activity levels in the optic tectum in a global rather than a site-specific manner.