PURPOSE. The purpose of this study was to characterize the ability of applied electrical fields (EFs) to direct retinal ganglion cell (RGC) axon growth as well as to assess whether Rho GTPases play a role in translating electrical cues to directional cues. METHODS. Full-thickness, early postnatal mouse retina was cultured in electrotaxis chambers and exposed to EFs of varying strengths (50-200 mV/mm). The direction of RGC axon growth was quantified from time-lapsed videos. The rate of axon growth and responsiveness to changes in EF polarity were also assessed. The effect of toxin B, a broad-spectrum inhibitor of Rho GTPase signaling, and Z62954982, a selective inhibitor of Rac1, on EF-directed growth was determined. RESULTS. In the absence of an EF, RGC axons demonstrated indiscriminate directional growth from the explant edge. Retinal cultures exposed to an EF of 100 and 200 mV/mm showed markedly asymmetric growth, with 74.2% and 81.2% of axons oriented toward the cathode, respectively (P < 0.001). RGC axons responded to acute changes in EF polarity by redirecting their growth toward the "new'' cathode. This galvanotropic effect was partially neutralized by toxin B and Rac1 inhibitor Z62954982. CONCLUSIONS. RGC axons exhibit cathode-directed growth in the presence of an EF. This effect is mediated in part by the Rho GTPase signaling cascade.