Synapse formation is thought to be initiated and regulated by the transsynaptic interaction of synaptic adhesion molecules, such as N-cadherin. However, relatively little is known about the functional role of N-cadherin in synapse formation, stabilization and plasticity. We have now used a live imaging approach to study presynaptic vesicle accumulations in N-cadherin-deficient, ES cell-derived neurons. To fluorescently label presynaptic vesicles, cultured ES cell-derived neurons were acutely transfected with a DsRed-VAMP2 fusion protein using the lipofectamin technique. Control experiments using co-transfection of DsRed-VAMP2 and EGFP-PSD95 revealed that almost all dendritic DsRed-VAMP2 puncta were associated with PSD95 and thus represent glutamatergic synapses. In homozygous N-cadherin-deficient, ES cell-derived neurons the density of dendritic presynaptic vesicle accumulations was similar as compared to heterozygous controls. We further analysed the size (area) of the DsRed-VAMP2 puncta. In immature neurons we observed large clusters of DsRed-VAMP2 puncta in N-cadherin-deficient neurons, suggesting that N-cadherin controls the lateral bordering of the vesicle pool. In more mature neurons no significant difference in area was detectable. In summary, our results indicate that N-cadherin is dispensable for synapse formation per se, while it appears to play an important role in limiting the lateral extent of the presynaptic vesicle pool.