Despite extensive work in the field of glioblastoma research no significant increase in survival rates for this devastating disease has been achieved. It is known that disturbance of intracellular Ca2+ ([Ca 2+]i) and intracellular pH (pHi) regulation could be involved in tumor formation. The sarco(endo)plasmic reticulum Ca 2+-ATPase (SERCA) is a major regulator of [Ca2+] i. We have investigated the effect of inhibition of SERCA by thapsigargin (TG) on [Ca2+]i and pHi in human primary glioblastoma multiforme (GBM) cells and GBM cell lines, compared with normal human astrocytes, using the fluorescent indicators fura-2 and BCECF, respectively. Basal [Ca2+]i was higher in SK-MG-1 and U87 MG but not in human primary GBM cells compared with normal astrocytes. However, in tumor cells, TG evoked a much larger and faster [Ca2+]i increase than in normal astrocytes. This increase was prevented in nominally Ca2+-free buffer and by 2-APB, an inhibitor of store-operated Ca 2+ channels. In addition, TG-activated Ca2+ influx, which was sensitive to 2-APB, was higher in all tumor cell lines and primary GBM cells compared with normal astrocytes. The pHi was also elevated in tumor cells compared with normal astrocytes. TG caused acidification of both normal and all GBM cells, but in the tumor cells, this acidification was followed by an amiloride- and 5-(N,N-hexamethylene)-amiloride-sensitive recovery, indicating involvement of a Na+/H+ exchanger. In summary, inhibition of SERCA function revealed a significant divergence in intracellular Ca 2+ homeostasis and pH regulation in tumor cells compared with normal human astrocytes. Copyright © 2005 the American Physiological Society.