Abstract: Activation of immediate early gene expression is a key event in stress-induced neuronal cell injury. To study whether changes in cytoplasmic calcium activity are necessary to activate neuronal immediate early gene expression, endoplasmic reticulum (ER) calcium stores of primary neurons were depleted by exposing cells to thapsigargin (Tg), an irreversible inhibitor of ER Ca2+-ATPase. Tg-induced rise in [Ca2+]i and the effect of loading neurons with the cell-permeable calcium chelator BAPTA-AM on this increase in [Ca2+]i were measured in fura-2-loaded cells by fluorescence microscopy. Changes in c-fos mRNA levels were evaluated by quantitative PCR. Tg treatment of neurons produced a pronounced rise in c-fos mRNA levels (∼10-fold more than DMSO) which peaked at 1 h after exposure. The Tg-induced rise in c-fos mRNA content was unchanged (hippocampal neurons) or even increased further (cortical neurons) by preloading cells with BAPTA before incubation with Tg. It is concluded that in neuronal cells an increase in cytoplasmic calcium activity is not a prerequisite for a rise in mRNA levels of c-fos. Thus, stress-induced changes in mRNA levels of immediate early genes of neurons may also result from disturbances in ER calcium homeostasis and not necessarily by an overload of cells with calcium ions. The results of the present series of experiments cast further doubt on the widely accepted hypothesis that the stress-induced cytoplasmic overload of neurons with calcium ions is the primary event triggering cell injury.