The possibility that eye enucleation within one day after birth has an effect on the postnatal development of substance P (SP)-like-immunoreactive (SP-I) structures in the superior colliculus (SC) was investigated in the rat. Results were compared with those in animals enucleated at postnatal day 15. All the animals were allowed to survive until postnatal day 90, after which changes in SP-I neurons and fibers were identified immunohistochemically. In colchicine-treated rats, the most remarkable changes occurred in SP-I neurons following eye enucleation at birth; large numbers of SP-I neurons appeared in the ventral part of the stratum griseum superficiale (SGS), stratum opticum (SO) and stratum griseum intermediale (SGI) of the deafferentated SC. SP-I neurons did not appear in these layers, when deafferentation of the SC was carried out in rats at postnatal day 15. These findings suggest strongly that eye enucleation at birth affects the production of SP of neurons in the ventral part of the SGS, SO and SGI at the deafferentated SC. The appearance of SP-I neurons in the neonatal eye enucleation may be due, at least partially, to reorganization of another neuronal system in the SC.