The inner membrane of the nuclear envelope (NE) was previously shown to contain a Na/Ca exchanger (NCX) tightly linked to GM1 ganglioside that mediates transfer of nucleoplasmic Ca²⁺ to the NE lumen and constitutes a cytoprotective mechanism. This transfer was initially observed with isolated nuclei and is now demonstrated in living cells in relation to subcellular Ca²⁺ dynamics. Four cell lines with varying expression of NCX and GM1 in the NE were transfected with cameleon-fluorescent Ca²⁺ indicators genetically targeted to NE/endoplasmic reticulum (ER) and nucleoplasm to monitor [Ca²⁺]_ne/er and [Ca²⁺]_n respectively. Cytosolic Ca²⁺ ([Ca²⁺]_cyt) was indicated with fura-2. Thapsigargin caused progressive loss of [Ca²⁺]_ne/er, which was rapidly replaced on addition of extrinsic Ca²⁺ to those cells containing fully functional NCX/GM1: differentiated NG108–15 and C6 cells. Reduced elevation of [Ca²⁺]_ne/er following thapsigargin depletion occurred in cells containing little or no GM1 in the NE: undifferentiated NG108–15 and NG-CR72 cells. No change in [Ca²⁺]_ne/er due to applied Ca²⁺ was seen in Jurkat cells, which entirely lack NCX. Ca²⁺ entry to NE/ER was also blocked by KB-R7943, inhibitor of NCX. [Ca²⁺]_n and [Ca²⁺]_cyt were elevated independent of [Ca²⁺]_ne/er and remained in approximate equilibrium with each other. Ca²⁺ rise in the ER originated in the NE region and extended to the entire ER network. These results indicate the nuclear NCX/GM1 complex acts to gate Ca²⁺ transfer from cytosol to ER, an alternate route to the sarcoplasmic/endoplasmic reticulum calcium ATPase pump. They also suggest a possible contributory mechanism for independent regulation of nuclear Ca²⁺.