Activity-dependent synaptic plasticity is characteristic of developing visual systems. Using the frog retinotectal system, we investigated the extent to which afferent input affects neurotransmitter expression in a target structure. We have concentrated on a particular subpopulation of tectal cells that is immunoreactive to substance P (SP). Early in development, SP expression in tectal neurons was restricted to the anterior lateral region of the tectum. As tadpoles developed, this expression expanded into progressively more posterior and medial regions in a manner that closely followed the gradient of tectal maturation. At all times, however, anterior and lateral tectal regions had a greater percent of SP-like immunoreactive (SP-ir) cells than posterior and medial ones. Horseradish peroxidase (HRP) labeling of the retinal ganglion cell projection in conjunction with SP immunocytochemistry demonstrated that innervation by retinal ganglion cell terminals preceded the expression of SP by tectal cells. This suggested that the optic nerve may influence SP differentiation and/or expression. In support of this idea, transection of the optic nerve resulted in a decrease in SP expression in the deafferented tectal lobe of tadpoles. This result, opposite to that seen previously in the adult, also indicates that optic nerve–dependent regulation of SP expression in the developing and mature systems occurs through different pathways.