In the classical view, NMDA receptors (NMDARs) are located postsynaptically and play a pivotal role in excitatory transmission and synaptic plasticity. In developing cerebellar molecular layer interneurons (MLIs) however, NMDARs are known to be solely extra- or presynaptic and somewhat poorly expressed. Somatodendritic NMDARs are exclusively activated by glutamate spillover from adjacent synapses, but the mode of activation of axonal NMDARs remains unclear. Our data suggest that a volume transmission is likely to stimulate presynaptic NMDARs (preNMDARs) since NMDA puffs directed to the axon led to inward currents and Ca2+ transients restricted to axonal varicosities. Using local glutamate photoliberation, we show that pre- and post-synaptic NMDARs share the same voltage dependence indicating their containing NR2A/B subunits. Ca2+ transients elicited by NMDA puffs are eventually followed by delayed events reminding of the spontaneous Ca2+ transients (ScaTs) described at the basket cell/Purkinje cell terminals. Moreover, the presence of Ca2+ transients at varicosities located more than 5 μm away from the uncaging site indicates that the activation of preNMDARs sensitizes the Ca2+ stores in adjacent varicosities, a process that is abolished in the presence of a high concentration of ryanodine. Altogether, the data demonstrate that preNMDARs act as high-gain glutamate detectors.