Ocular dominance columns in the cat's visual cortex appear to develop out of an initially overlapping projection by a progressive segregation of the geniculocortical afferents serving the 2 eyes (reviewed in LeVay and Stryker, 1979). To determine whether electrical activity in the visual afferent pathway is involved in this normal, developmental rearrangement of synaptic connections, we blocked the discharge of retinal ganglion cells in both eyes by making repeated intravitreal injections of tetrodotoxin (TTX) during the period in which geniculocortical afferent segregation would normally be taking place. Control experiments for the side effects of the injection procedure, the systemic effects of TTX, and the effects of visual deprivation were carried out, and a series of normal animals of appropriate ages was also studied. We then examined the effects of retinal blockade and the various control procedures on the formation of ocular dominance columns using an anatomical assay, the autoradiographic labeling of geniculocortical afferent terminals in layer IV of the visual cortex by the transneuronal transport of tritiated proline injected into 1 eye, and a physiological assay, the ocular dominance of single cortical cells recorded extracellularly. After retinal TTX blockade, layer IV was labeled uniformly without periodic fluctuation in grain density, and nearly all cortical cells were driven well through both eyes. These assays thus indicated that retinal blockade completely blocked the formation of ocular dominance columns, unlike any of the control procedures, suggesting that the spontaneous maintained discharge of retinal ganglion cells may have an important role in the normal development of binocular connections in the visual cortex.