Substance-P-like immunoreactivity (SP-LI) cells in the Long-Evans rat retina were investigated by combining immunohistochemistry with [3H]thymidine autoradiography. Two subpopulations of SP-LI amacrine cells, with cell bodies in either the proximal portion of the inner nuclear layer (INL) or the ganglion cell layer (GCL), were identified based on morphology, pattern of distribution and development. In the INL, SP-LI cells were found scattered throughout the retina. However, in the GCL, they were limited to the superio-temporal region. Such a contrast in distribution specific to nuclear layers was present upon first detection of SP-LI amacrine cells and persisted throughout development. Birthdating revealed a temporal lag in the histogenesis of SP-LI cells situated in the GCL relative to that in the INL, suggesting that the two subpopulations developed separately. Overall, unique anatomical features of the SP-LI amacrine cells in the rat retina were observed which could only have been uncovered through detailed analyses in the adult as well as during postnatal development.