Synaptic transmission is maintained by a delicate, subsynaptic molecular architecture, and even mild alterations in synapse structure drive functional changes during experience-dependent plasticity and pathological disorder^1,2. Key to this architecture is how the distribution of presynaptic vesicle fusion sites corresponds to the position of receptors in the postsynaptic density. However, despite long recognition that this spatial relationship modulates synaptic strength³, it has not been precisely described, due in part to the limited resolution of light microscopy. Using localization microscopy, we report here that key proteins mediating vesicle priming and fusion are mutually co-enriched within nanometer-scaled subregions of the presynaptic active zone. Through development of a new method to map vesicle fusion positions within single synapses, we found that action potential evoked fusion was guided by this protein gradient and occurred preferentially in confined areas with higher local density of RIM within the active zones. These presynaptic RIM nanoclusters closely aligned with concentrated postsynaptic receptors and scaffolding proteins^4–6, suggesting a transsynaptic molecular “nanocolumn.” Thus, we propose that the nanoarchitecture of the active zone directs action potential evoked vesicle fusion to occur preferentially at sites directly opposing postsynaptic receptor-scaffold ensembles. Remarkably, NMDA receptor activation triggered distinct phases of plasticity in which postsynaptic reorganization was followed by transsynaptic nanoscale realignment. This architecture thus suggests a simple organizational principle of CNS synapses to maintain and modulate synaptic efficiency.