Substance P-like immunoreactivity (SPLI) was immunohistochemically analyzed in the retinae and optic tecta of Rana pipiens embryos and tadpoles between stages 25 of Shumway (S25) and XXV of Taylor and Kollros (TKXXV). A population of retinal ganglion cell (RGC) somata display SPLI. The number of labeled cell bodies increases in proportion and staining intensity between S25 and TKX and progressively decreases toward the end of metamorphosis. At TKXXV, only occasional cells in the periphery of the retina displaying SPLI can be observed in the RGC layer, heralding the adult condition, in which SPLI can only be seen rarely in occasional RGCs. An increasing proportion of optic nerve axons display SPLI from S25 through TKXVI, decreasing progressively thereafter toward the end of the larval period. Concurrently, SPLI appears for the first time in the superficial tectal neuropil between TKIII and TKV, with progressively increasing staining intensity and in a discrete lamina previously shown to contain retinofugal terminals in the adult. These observations corroborate inferences from previous studies indicating the existence of populations of peptidergic RGCs that terminate within precisely restricted synaptic loci in the tectum and presumably perform different functional operations in the adult. Previous observations, however, necessitated various experimental manipulations involving injuries to the visual system in order to demonstrate neuroactive peptide-like immunoreactivity in RGCs, thus allowing the possibility of posttraumatic expression of anomalous peptide phenotypes that may not reflect normal features of RGCs. The present study eliminates this variable and provides further evidence of the existence of peptidergic RGCs.