Elevation of calcium during sustained synaptic activity may lead to the activation of the postsynaptic calcium-dependent protease calpain and thus could alter the integrity and localization of endogenous proteins. The distribution of anchoring proteins for neuroreceptors is an important determinant of the efficacy of neuronal transmission. Many of these anchoring proteins are concentrated within the postsynaptic density (PSD). In the present study, we examined the effects of calpain II on isolated PSDs using biochemical and electron microscopic techniques. Biochemical analysis reveals that PSD-95, a clustering molecule which anchors NMDA receptors by interaction with their NR2 subunits, as well as the NR2 subunits themselves, are cleaved by calpain. On the other hand, under conditions where all the PSD-95 protein is cleaved, actin and alpha-actinin-a protein thought to anchor NMDA receptors to actin filaments-remain intact. For analysis by electron microscopy, PSDs were adsorbed on glass, immunogold-labeled with an antibody to PSD-95, slam frozen, freeze dried, and rotary shadowed. Electron micrographs of replicas indicate that PSDs are disc-shaped and are composed of a lattice-like structure which labels with PSD-95 immunogold. After calpain treatment, PSDs adsorbed on glass become thinner overall and the lattice becomes fragmented. Altogether, these results suggest that calpain activity could produce changes in the organization of the PSD and, by cleaving PSD-95 associated with the PSD lattice, could modify the anchoring of NMDA receptors.