The polyether antibiotic ionomycin is a common research tool employed to raise cytosolic Ca(2+) in almost any cell type. Although initially thought to directly cause physicochemical translocation of extracellular Ca(2+) into the cytosol, a number of studies have demonstrated that the mechanism of action is likely to be more complex, involving modulation of intrinsic Ca(2+) signaling pathways. In the present study we assessed the effect of ionomycin on primary cultures of murine cerebellar astrocytes. Ionomycin concentrations ranging from 0.1 to 10 µM triggered a biphasic increase in cytosolic Ca(2+), consisting of an initial peak and a subsequent sustained plateau. The response was dependent on concentration and exposure time. While the plateau phase was abolished in the absence of extracellular Ca(2+), the peak phase persisted. The peak amplitude could be lowered significantly by application of dantrolene, demonstrating involvement of Ca(2+)-induced Ca(2+)-release (CICR). The plateau phase was markedly reduced when store-operated Ca(2+)-entry (SOCE) was blocked with 2-aminoethoxydiphenyl borate. Our results show that ionomycin directly affects internal Ca(2+) stores in astrocytes, causing release of Ca(2+) into the cytosol, which in turn triggers further depletion of the stores through CICR and subsequently activates SOCE. This mechanistic action of ionomycin is important to keep in mind when employing it as a pharmacological tool.