The glucose-regulated protein grp78 gene is rapidly transactivated in 9L rat brain tumour (RBT) cells treated with okadaic acid (OA) followed by heat shock (HS) (termed OA→HS treatment). By Northern blotting analyses and transient transfection assays, we herein show that transactivation of grp78 by OA→HS is abolished by an intracellular calcium chelator, bis(aminophenoxy)ethane N,N′-tetraacetic acid (BAPTA), and an inhibitor of mitochondrial Ca2+ uniporter, ruthenium red (RR), while unaffected by cyclosporin A (CsA), an inhibitor of mitochondrial permeability transition pore (MTP). The inhibitory effects of BAPTA and RR also present in OA→HS induction of transient elevation of intracellular hydrogen peroxide. The requirement of reactive oxygen intermediates (ROIs) is confirmed by substitutional addition of antioxidants, N-acetyl cysteine (NAC) and pyrrolidinedithiocarbamate (PDTC) during OA→HS treatment, mimicking these inhibitory effects of BAPTA and RR. Western blotting analyses show that phosphorylation of transcription factor CREB is diminished only by BAPTA but not by RR, while phosphorylation of ATF-2 is unaffected by either agent. Conclusively, we present that both the disturbances of mitochondrial calcium homeostasis and reactive oxygen intermediates are essential for rapid transactivation of grp78, and this pathway is separate from protein kinase A (PKA)-dependent CREB activation or p38 mitogen-activated protein kinase (p38MAPK)-dependent ATF-2 activation and signalling.