The formation of a retinotopic map is thought to involve an activity-independent molecular phase for early steps of both axon pathfinding and projection and a later phase in which cross talk between retinal ganglion cells (RGCs) and tectal neurons modifies and refines the neuronal connections. We report that the maturation of the retinotopic map in the zebrafish tectum involves activity-dependent processes. Zebrafish larvae mutant for the gene macho (mao) lack neuronal activity in RGCs and also display an enlarged retinotectal projection field but no significant increase in single axon length. This morphological defect can be phenocopied by raising larvae under TTX-induced neural impulse blockade. The effect of activity deprivation is dependent on the developmental stage. The projection phenotype in mao as well as in the TTX-treated larvae develops between 4 and 6 d post-fertilization (dpf), after complete tectal coverage is first achieved. Electrophysiological recordings of RGCs in wild-type and mao zebrafish larvae reveal a temporally regulated reduction of sodium current in the mutant between 5 and 6 dpf. This coincides with the time of the axonal projection shifting on the tectum to compensate for the disparate growth patterns of the retina and the tectum. Our genetic and physiological analyses suggest a model in which neuronal activity in RGCs is needed for the establishment of morphological plasticity.