In the mammalian visual system, retinal ganglion cell (RGC) projections from each eye, initially intermixed within the dorsal-lateral geniculate nucleus (dLGN), become segregated during the early stages of development, occupying distinct eye-specific layers. Electrical activity has been suggested to play a role in this process; however, the cellular mechanisms underlying eye-specific segregation are not yet defined. It is known that electrical activity is among the strongest activators of the extracellular signal-regulated kinase (ERK) pathway. Moreover, the ERK pathway is involved in the plasticity of neural connections during development. We examine the role of ERK in the segregation of retinal afferents into eye-specific layers in the dLGN. The activation of this signaling cascade was selectively blocked along the retino-thalamic circuitry by specific inhibitors, and the distribution of RGC fibers in the dLGN was studied. Our results demonstrate that the blockade of ERK signaling prevents eye-specific segregation in the dLGN, providing evidence that ERK pathway is required for the proper development of retino-geniculate connections. Of particular interest is the finding that ERK mediates this process both at the retinal and geniculate level.