Synaptic adhesion molecules such as N-cadherin and Neuroligin1 play major roles in regulating synapse structure and function. At the molecular level, several different families of synaptic adhesion molecules have been described to mediate trans-synaptic signaling. This led to the proposal of a complex co-operation of different types of adhesion molecules that together might control synapse formation. Using N-cadherin knockout neurons derived from mouse ES cells, we found by immunocytochemistry, live-cell imaging of individual vesicle clusters, and FRAP experiments that N-cadherin is strongly involved in the accumulation of synaptic vesicles at nascent synapses. Intriguingly, N-cadherin appears to control vesicle accumulation indirectly, because N-cadherin expression alone does not induce presynaptic vesicle clusters. To further address this, we studied the accumulation of presynaptic vesicles that is induced by Neuroligin1 expression in cultured N-cadherin knockout neurons. Strikingly, in the absence of N-cadherin expression Neuroligin1 did not induce additional vesicle clustering. We confirmed this finding in CA3 pyramidal neurons in organotypic hippopcampal slice cultures. Blocking N-cadherin function abolished the vesicle clustering activity of transiently expressed Neuroligin1. In addition to its vesicle clustering activity also the synaptic targeting of expressed Neuroligin1 was impaired in the absence of N-cadherin. Most importantly, also the synaptic clustering of endogeneous Neuroligin was reduced in N-cadherin knockout neurons. The N-cadherin and Neuroligin adhesion systems appeared to be linked by postsynaptic scaffolding proteins as indicated by experiments impeding S-SCAM function. In summary, our data support a molecular co-operation model, in which N-cadherin might indirectly control presynaptic vesicle accumulation by targeting and activating postsynaptic Neuroligin1, which ultimately induces presynaptic vesicle clustering via neurexin binding.