Current models of retinogeniculate development have proposed that connectivity between the retina and the dorsal lateral geniculate nucleus (dLGN) is established by gradients of axon guidance molecules, to allow initial coarse connections, and by competitive Hebbianlike processes, to drive eye-specific segregation and refine retinotopy. Here we show that when intereye competition is eliminated by monocular enucleation, blocking cholinergic stage II retinal waves disrupts the intraeye competition-mediated expansion of the retinogeniculate projection and results in the permanent disorganization of its laminae. This disruption of stage II retinal waves also causes longterm impacts on receptive field size and fine-scale retinotopy in the dLGN. Our results reveal a novel role for stage II retinal waves in regulating retinogeniculate afferent terminal targeting by way of intraeye competition, allowing for correct laminar patterning and the even allocation of synaptic territory. These findings should contribute to answering questions regarding the role of neural activity in guiding the establishment of neural circuits.retinal waves | retinogeniculate | axon-axon competition | receptive fields | retinotopy T he brain employs several strategies to guide the establishment of correct neural connectivity (1, 2). It has been well recognized that the high specificity of connections between the retina and the dorsal lateral geniculate nucleus (dLGN) is established through several factors. These include gradients of axon guidance molecules that guide the initial coarse targeting of afferent terminals (3-6), and spontaneous retinal activity (retinal waves) that drives competitive processes important for the refinement and segregation of afferent terminal branches (2, 7-15).Retinal waves are spontaneous propagating bursts of correlated retinal ganglion cell (RGC) activity and have been classified into three developmental stages (1, 15). Stage II retinal waves (from here on also referred to as retinal waves) are extensively studied and have been found to be critical for the development of retinofugal pathways (1, 2, 15). They are mediated by cholinergic signaling from starburst amacrine cells onto RGCs (8,13,[16][17][18] and have been hypothesized to drive the Hebbian-like remodeling of RGC afferent terminals (19,20). Retinal waves play crucial roles in both the establishment of eye-specific segregation (8,12,14,20,21), through the removal of afferent branches from opposing putative eyespecific domains, and the refinement of afferent terminals within eye-specific laminae, which is believed to be necessary for the establishment of fine-scale retinotopy (12,22). However, studies have suggested that retinal waves might play additional roles in the development of the retinogeniculate pathway. When retinal waves are blocked during early development, mature lamination in the adult is abnormal (23-25), while eye-specific segregation recovers (26,27). These results uncovered a retinal wave-dependent window for the development of retinogen...