The mechanism of action of the oncogene bcl-2, a key regulator of the apoptotic process, is still debated. We have employed organelle-targeted chimeras of the Ca²⁺-sensitive photoprotein, aequorin, to investigate in detail the effect of Bcl-2 overexpression on intracellular Ca²⁺ homeostasis. In the ER and the Golgi apparatus, Bcl-2 overexpression increases the Ca²⁺ leak (while leaving Ca²⁺ accumulation unaffected), hence reducing the steady-state [Ca²⁺] levels. As a direct consequence, the [Ca²⁺] increases caused by inositol 1,4,5 trisphosphate (IP3)-generating agonists were reduced in amplitude in both the cytosol and the mitochondria. Bcl-2 overexpression also reduced the rate of Ca²⁺ influx activated by Ca²⁺ store depletion, possibly by an adaptive downregulation of this pathway. By interfering with Ca²⁺-dependent events at multiple intracellular sites, these effects of Bcl-2 on intracellular Ca²⁺ homeostasis may contribute to the protective role of this oncogene against programed cell death.