In spite of 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 calcium ([Ca²⁺]_i) and pH (pH_i) regulation could be involved in tumor formation. The endoplasmic reticulum Ca²⁺-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 pH_i in human primary glioblastoma (GBM) cells and GBM cell lines, as compared to 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 as compared to 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 calcium channels). Additionally, TG-activated Ca²⁺ influx, which was sensitive to 2-APB, was higher in all tumor cell lines and primary GBM cells as compared to normal astrocytes. The pH_i was also elevated in tumor cells compared to 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 calcium homeostasis and pH regulation in tumor cells as compared to normal human astrocytes.