The synaptic cell adhesion molecule neuroligin bound to beads has been shown to induce the clustering of neurxin and synaptic vesicle proteins in contacting axons . In addition, neurexin-coated beads induced clustering of neuroligin and postsynaptic proteins in contacting dendrites . We employed this assay to demonstrate that the extracellular domain of NGL, a family of cell adhesion molecules that associates with the netrin-G family of cell adhesion molecules and the postsynaptic scaffolding protein PSD-95, bound to beads induces presynaptic differentiation in contacting axons of cultured neurons. Presynaptic differentiation was visualized by immunostaining for the presynaptic vesicle protein synaptophysin and vesicular glutamate transporter 1 (VGlut1; a marker for excitatory presynapses). In addition, functional presynaptic differentiation was demonstrated by vesicle turnover experiments, which monitor the uptake of synaptotagmin luminal domain antibodies .