The modulation of Ca2+ signaling patterns during repetitive stimulations represents an important mechanism for integrating through time the inputs received by a cell. By either overexpressing the isoforms of protein kinase C (PKC) or inhibiting them with specific blockers, we investigated the role of this family of proteins in regulating the dynamic interplay of the intracellular Ca2+ pools. The effects of the different isoforms spanned from the reduction of ER Ca2+ release (PKCalpha) to the increase or reduction of mitochondrial Ca2+ uptake (PKCzeta and PKCbeta/PKCdelta, respectively). This PKC-dependent regulatory mechanism underlies the process of mitochondrial Ca2+ desensitization, which in turn modulates cellular responses (e.g., insulin secretion). These results demonstrate that organelle Ca2+ homeostasis (and in particular mitochondrial processing of Ca2+ signals) is tuned through the wide molecular repertoire of intracellular Ca2+ transducers.