During vertebrate neuromuscular junction (NMJ) development, presynaptic motor axons differentiate into nerve termini enriched in synaptic vesicles (SVs). At the nerve terminal, mitochondria are also concentrated, but how mitochondria become localized at these specialized domains is poorly understood. This process was studied in cultured Xenopus spinal neurons with mitochondrion-specific probe MitoTracker and SV markers. In nerve-muscle cocultures, mitochondria were concentrated stably at sites where neurites and muscle cells formed NMJs, and mitochondria coclustered with SVs where neurites were focally stimulated by beads coated with growth factors. Labeling with a mitochondrial membrane potential-dependent probe JC-1 revealed that these synaptic mitochondria were with higher membrane potential than the extrasynaptic ones. At early stages of bead-stimulation, actin-based protrusions and microtubule fragmentation were observed in neurites at bead contact sites, suggesting the involvement of cytoskeletal dynamics and rearrangement during presynaptic differentiation. Treating the cultures with an actin polymerization blocker, latrunculin A (Ltn A), almost completely abolished the formation of actin-based protrusions and partially inhibited bead-induced mitochondrial and SV clustering, whereas the microtubule disrupting agent nocodazole was ineffective in inhibiting the clustering of mitochondria and SVs. Lastly, in contrast to Ltn A, which blocked bead-induced clustering of both mitochondria and SVs, the ser/thr phosphatase inhibitor okadaic acid inhibited SV clustering but not mitochondrial clustering. These results suggest that at developing NMJs, synaptogenic stimuli induce the clustering of mitochondria together with SVs at presynaptic terminals in an actin cytoskeleton-dependent manner and involving different intracellular signaling molecules.