Previously we demonstrated that the tectal gradient of EphA3 participates in the retinotectal mapping by stimulating the axon growth of nasal retinal ganglion cells (RGC) towards the caudal tectum and inhibiting their branching in the rostral tectum. This process is mediated by a decrease in ephrin-A-dependent activity of axonal EphA4. Eph/ephrin interactions take place in the cholesterol-rich lipid rafts. There are conflictive data about the role of colesterol microenvironment on axonal growth and guidance and the dynamics of this process is incompletelly understood. Our objective was to characterize the dynamics of nasal RGC axon growth and guidance mediated by EphA3 gradient and determine the relationship of the cholesterol microenvironment with this process. For this purpose, dissociated NRGCs from chicken embryos were cultured and exposed to a gradient of EphA3-Fc produced in the Dunn’s chemotaxis chamber in time lapse. We also evaluated the effect of depletion of the plasma membrane cholesterol with βMCD. The results showed that the EphA3 gradient has a positive chemotactic effect on NRGC by increasing the axon extension rate and promoting directional attraction, effects that were significantly decreased by transient cholesterol depletion. Our findings provide novel insights into how axon dynamics are regulated by EphA3 during retinotectal map formation and demonstrate that the cholesterol microenvironment modulates this process. Support: UBACYT0197BA.