Retrograde signals from axon terminal to cell body are known to regulate neuronal survival and differentiation during development. They are generally attributed to the uptake and transport of trophic factors, but there is recent evidence in the isthmo-optic nucleus for a remarkably fast-acting retrograde signal from the contralateral retina that is not mediated by the conventional trophic route. The isthmo-optic nucleus undergoes 55% neuron death between embryonic days 12 and 17, and becomes laminated at embryonic day 14 owing to dendritic re-organization. Blockade of retinal electrical activity just before day 14 reduces neuronal death and lamination in the isthmo-optic nucleus within as little as 6 h. We here investigate how action potentials initiate the fast-acting retrograde signal, and we provide evidence that the first step is calcium entry into the isthmo-optic axon terminals. Neuronal death and lamination are rapidly reduced in the isthmo-optic nucleus by intraocularly injected omega-conotoxin, a blocker of N-type calcium channels known to be located mainly on axon terminal. Similar effects occurred with two other calcium channel blockers (cadmium and alpha-bungarotoxin) believed to act on both the isthmo-optic terminals and their target cells, but not with nifedipine, a blocker of L-type (mainly somatic) channels, supporting a presynaptic initiation of the fast signal. Nevertheless postsynaptic events may also be involved because pharmacological destruction of the amacrine targets cells of the isthmo-optic nucleus reduced its cell death and lamination 9-12 h later.