Axon pathfinding requires directional responses of growth cones to extracellular cues, which have been shown to involve local synthesis of protein. The identity and functions of the locally produced proteins remain, however, unclear. Here we report that Ca(2+)-dependent bidirectional turning of Xenopus laevis growth cones requires localized distribution and translation of beta-actin messenger RNA. Both beta-actin mRNA and its zipcode-binding protein, ZBP1, are localized at the growth cone and become asymmetrically distributed upon local exposure to brain-derived neurotrophic factor (BDNF). Inhibition of protein synthesis or antisense interference with beta-actin mRNA-ZBP1 binding abolishes both Ca(2+)-mediated attraction and repulsion. In addition, attraction involves a local increase in beta-actin, whereas repulsion is accompanied by a local decrease in beta-actin; thus, both produce a synthesis- and ZBP1 binding-dependent beta-actin asymmetry but with opposite polarities. Together with a similar asymmetry in Src activity during bidirectional responses, our findings indicate that Ca(2+)-dependent spatial regulation of beta-actin synthesis through Src contributes to the directional motility of growth cones during guidance.