We have performed simultaneous real time recording of the intracellular level of Ca2+ ([Ca2+]i) and intracellular localization of protein kinase C (PKC) in cultured rat cortical neurons. The neurons were transfected with a plasmid that encoded a chimerical protein of PKCβII and green fluorescent protein (GFP) and loaded with fura-2FF. In the resting neurons, PKCβII-GFP was uniformly distributed in the cytoplasm of cortical neurons. The primary [Ca2+]i increase caused by glutamate was accompanied by translocation of PKCβII-GFP from the cytosol to the plasma membrane. A secondary [Ca2+]i elevation (destabilization of calcium homeostasis) was associated with retranslocation of PKCβII-GFP from the plasma membrane to the cytoplasmic organelle-like structures. Similar organelle-like structures were observed after immunohistochemical staining of PKCβII in cultured cerebellar granule cells and cortical neurons subjected to long-term glutamate treatment. It seems that PKCβII was active in these structures because the receptor for activated C kinase RACK1 also translocated into similar structures. Formation of clusters by PKCβII was calcium-dependent because the clusters also formed after treatment of neurons with calcium ionophore ionomycin. No retranslocation of PKC was observed after treatment with the calcium-independent activator of PKCβII phorbol-myristate acetate (PMA); in this case both PKCβII and RACK1 remained on the plasma membrane of neurons. The results obtained suggest that deregulation of neuronal calcium homeostasis during hyperstimulation of glutamate receptors induces specific changes in the localization of active PKCβII.