Presynaptic NMDA receptors ( NMDARs) modulate release and plasticity at many glutamatergic synapses, but the specificity of their expression across synapse classes has not been examined. We found that non-postsynaptic, likely presynaptic NR2B-containing NMDARs enhanced AMPA receptor-mediated synaptic transmission at layer 4 ( L4) to L2/3 ( L4-L2/3) synapses in juvenile rat barrel cortex. This modulation was apparent at room temperature when presynaptic NMDARs were activated by elevation of extracellular glutamate or application of exogenous NMDAR agonists. At near physiological temperatures, modulation of transmission by presynaptic NMDARs occurred naturally, without the need for external activation. Blockade of presynaptic NMDARs depressed unitary and extracellularly evoked EPSCs at L4 -L2/3 synapses, accompanied by increases in paired-pulse ratio and coefficient of variation, indicative of a decrease in presynaptic release probability. NMDAR agonists increased the frequency of miniature EPSCs in L2/3 neurons, without altering their amplitude or kinetics. Focal application of NMDAR antagonist revealed that the NMDARs that modulate L4-L2/3 transmission are located in L2/3, not L4, consistent with localization on terminals or axons of L4-L2/3 synapses, rather than on the somato-dendritic compartment of presynaptic L4 neurons. In contrast, presynaptic NMDARs did not modulate L4 -L4 synapses, which originate from the same presynaptic neurons as L4-L2/3 synapses, or cross-columnar L2/3-L2/3 horizontal projections, which synapse onto the same postsynaptic target neurons. Thus, presynaptic NMDARs selectively modulate L4-L2/3 synapses, relative to other synapses made by the same neurons. Existence of these receptors may support specialized processing or plasticity by L4-L2/3 synapses.