1 SUMMARY Developing neural circuits are particularly vulnerable to alterations in early sensory experience. For example, monocular deprivation (MD) during a juvenile critical period leads to long-lasting changes in binocularity and spatial acuity of the visual system. The locus of these changes has been widely considered to be cortical. However, recent evidence indicates that binocular integration occurs first in the dorsolateral geniculate nucleus of the thalamus (dLGN) and that dLGN binocularity may be susceptible to changes in visual experience. This leaves open the question of whether MD during the critical period leads to long-lasting deficits in dLGN binocular integration. Using in vivo two-photon Ca 2+ imaging of dLGN afferents and excitatory neurons in superficial layers of binocular V1, we demonstrate that critical-period MD leads to a persistent and selective loss of binocular dLGN inputs, while leaving visual acuity in the thalamocortical pathway intact. Moreover, we found profound mismatch of preferred spatial frequency and orientation detected at the level of individual thalamocortical synapses. In V1 neurons, we found significant reductions in binocularity and visual acuity following critical-period MD. Our data suggest that alterations in cortical ocular dominance and binocular matching following critical-period MD may partially reflect a dysfunction of binocular integration in dLGN neurons.