Rab GTPases use the guanine nucleotide–dependent switch–mechanism to regulate membrane traffic. Rab35, which is localized at the plasma membrane, the cytosol and the internal vesicles, has been shown to be implicated in a variety of functions including cytokinesis, receptor recycling, and neurite outgrowth. Recently, it has been reported that Rab35 and Arf6 work together for the biogenesis of recycling endosome . On the other hands, Rab11 is key regulator of recycling/secretion pathways and are implicated in neurite outgrowth as well as Rab35. Thus, it is of immediate interest where and when Rab35 activity changes and how does the role of Rab35 intersect with that of Rab11. To answer these questions, we utilized a FRET sensor, Raichu–Rab35. In NGF–treated PC12 cells, neurite tips showed a higher Rab35 activity than that in the neurite shaft. Furthermore, we examined the dynamics of Rab35–containing vesicles in neurite–bearing N1E–115 cells. In shafts, Rab35–positive vesicles were transported both anterogradely and retrogradely. In growth cones, most of Rab35–containing vesicles resided in central domains and showed anterogradely movement. We frequently observed the vesicles containing both Rab35 and Rab11 in neurite shafts and growth cones. This study provides a clue to elucidate the promoting role of Rab35 in neurite outgrowth.