Zinc is hypothesized to be co-released with glutamate at synapses of the central nervous system. Zinc binds to NR1/NR2A N-methyl-d-aspartate (NMDA) receptors with high affinity and inhibits NMDAR function in a voltage-independent manner. The serine protease plasmin can cleave a number of substrates, including protease-activated receptors, and may play an important role in several disorders of the central nervous system, including ischemia and spinal cord injury. Here, we demonstrate that plasmin can cleave the native NR2A amino-terminal domain (NR2A(ATD)), removing the functional high affinity Zn(2+) binding site. Plasmin also cleaves recombinant NR2A(ATD) at lysine 317 (Lys(317)), thereby producing a approximately 40-kDa fragment, consistent with plasmin-induced NR2A cleavage fragments observed in rat brain membrane preparations. A homology model of the NR2A(ATD) predicts that Lys(317) is near the surface of the protein and is accessible to plasmin. Recombinant expression of NR2A with an amino-terminal deletion at Lys(317) is functional and Zn(2+) insensitive. Whole cell voltage-clamp recordings show that Zn(2+) inhibition of agonist-evoked NMDA receptor currents of NR1/NR2A-transfected HEK 293 cells and cultured cortical neurons is significantly reduced by plasmin treatment. Mutating the plasmin cleavage site Lys(317) on NR2A to alanine blocks the effect of plasmin on Zn(2+) inhibition. The relief of Zn(2+) inhibition by plasmin occurs in PAR1(-/-) cortical neurons and thus is independent of interaction with protease-activated receptors. These results suggest that plasmin can directly interact with NMDA receptors, and plasmin may increase NMDA receptor responses through disruption or removal of the amino-terminal domain and relief of Zn(2+) inhibition.