Although activation of calcium-activated neutral protease (calpain) by the NMDA receptor has been suggested to play critical roles in synaptic modulation and neurologic disease, the nature of its substrates has not been completely defined. In this study, we examined the ability of calpain to cleave the NMDA receptor in cultured hippocampal neurons. Activation of the NMDA receptor by agonist application led to rapid calpain-specific proteolysis of spectrin and decreased levels of NR2A/2B subunits. Cleavage of the NR2A/2B subunit created a 115 kDa product that retained the ability to bind ¹²⁵I-MK-801 and is predicted to be active. Increases in levels of this product appeared within 5 min of NMDA receptor activation and were stable for periods of >30 min. Subtype-specific antibodies demonstrated that the NR2B subunit was cleaved in these primary cultures, but the NR2A subunit was not. An inhibitor of calpain blocked both the decrease of intact NR2B and the increase of the low molecular weight form, whereas neither caspase nor cathepsin inhibitors had an effect on these events. Cell surface biotinylation experiments demonstrated that the 115 kDa fragment remained on the cell surface. This NR2B fragment was also found in the rat hippocampus after transient forebrain ischemia, showing that this process also occurs in vivo. This suggests that calpain-mediated cleavage of the NR2B subunit occurs in neurons and gives rise to active NMDA receptor forms present on the cell surface after excitotoxic glutamatergic stimulation. Such forms could contribute to excitotoxicity and synaptic remodeling.