In the adult mammalian visual system, ganglion cell axons from the two eyes are segregated from each other into separate layers within their principal target, the lateral geniculate nucleus. The involvement of spontaneously generated action potential activity in the process of segregation was investigated during the fetal period in which segregation normally occurs in the cat, between embryonic day 45 (E45) and birth (E65). Tetrodotoxin, which blocks the voltage-sensitive sodium channel, was used to prevent action potentials. Fetuses received continuous intracranial infusions of tetrodotoxin from osmotic minipumps implanted in utero on E42. After a 2-week infusion, intraocular injections of anterograde tracers revealed that tetrodotoxin prevented segregation. The contralateral projection filled the lateral geniculate nucleus uniformly, and the ipsilateral projection expanded to occupy most of what would normally be contralaterally innervated layer A. Thus, in the fetus, long before the onset of vision, spontaneous action potential activity is likely to be present in the visual system and to contribute to the segregation of the retinogeniculate pathway.