NMDA receptors (NMDARs) are required for experience-driven plasticity during formative periods of brain development and are critical for neurotransmission throughout postnatal life. Most NMDAR functions have been ascribed to postsynaptic sites of action, but there is now an appreciation that presynaptic NMDARs (preNMDARs) can modulate neurotransmitter release in many brain regions, including the neocortex. Despite these advances, the cellular mechanisms by which preNMDARs can affect neurotransmitter release are largely unknown. Here we interrogated preNMDAR functions pharmacologically to determine how these receptors promote spontaneous neurotransmitter release in mouse primary visual cortex. Our results provide three new insights into the mechanisms by which preNMDARs can function. First, preNMDARs can enhance spontaneous neurotransmitter release tonically with minimal extracellular Ca(2+) or with major sources of intracellular Ca(2+) blocked. Second, lowering extracellular Na(+) levels reduces the contribution of preNMDARs to spontaneous transmitter release significantly. Third, preNMDAR enhance transmitter release in part through protein kinase C signaling. These data demonstrate that preNMDARs can act through novel pathways to promote neurotransmitter release in the absence of action potentials.