1-(2-Chlorophenyl-N-methylpropyl)-3-isoquinolinecarboxamide (PK11195) is a proven enhancer of apoptotic cell death in a variety of cellular models. This effect is independent of its established cellular target, the mitochondrial benzodiazepine receptor (mBzR), since it is able to promote cell death also in mBzR knockout cells. Thus recently it was suggested that PK11195 might exert its effect by modulating the expression and function of the oncogene Bcl-2. We have previously demonstrated that Bcl-2 modulates cellular Ca2+ homeostasis as its overexpression reduces the Ca2+ concentration in the endoplasmic reticulum (ER) ([Ca2+](er)), impairing mitochondrial and cytosolic Ca2+ overload during cellular stress and therefore inhibiting the induction of the apoptotic cascade. Here, using ER, mitochondria and cytosolic targeted aequorin probes, we show that cellular treatment with PK11195 induces opposite changes in cellular Ca2+ homeostasis, increasing the [Ca2+](er) and amplifying IP(3) induced Ca2+ transients in mitochondria ([Ca2+](m)) and cytosol ([Ca2+](c)). This work provides evidence for a novel pharmacological effect of PK11195 on Ca2+ signalling which may be linked to its effect on Bcl-2 and account for its role in apoptotic cell death.