Spontaneous retinal activity mediated by cholinergic transmission regulates the segregation of retinal ganglion cell axons in the lateral geniculate nucleus of the thalamus into eye-specific layers. The details of how the layers form are unknown. Mice lacking the beta2 subunit of the neuronal nicotinic acetylcholine receptor lack ACh-mediated waves and as a result, do not form eye-specific layers at any stage of development. However, during the second postnatal week, beta2-/- mice have glutamate-mediated waves. Here we show that after the first postnatal week, even in the absence of layers, retinothalamic axons segregate into an unlayered, patchy distribution of eye-specific regions. These results indicate that spontaneous neural activity may independently regulate eye-specific segregation and the formation of layers at the developing retinothalamic projection.