We have previously demonstrated [M. Campos-Toimil, T. Bagrij, J.M. Edwardson, P. Thomas, Two modes of secretion in pancreatic acinar cells: involvement of phosphatidylinositol 3-kinase and regulation by capacitative Ca2+ entry, Curr. Biol. 12 (2002) 211–215] that in rat pancreatic acinar cells, Gd3+-sensitive Ca2+ entry is instrumental in governing which second messenger pathways control secretory activity. However, in those studies, we were unable to demonstrate a significant increase in cytoplasmic [Ca2+] during agonist application as a result of this entry pathway. In the present study, we combined pharmacology with ratiometric imaging of fura-2 fluorescence to resolve this issue. We found that 2 μM Gd3+ significantly inhibits store-mediated Ca2+ entry. Furthermore, both the protonophore, CCCP (5 μM) and the mitochondrial Ca2+-uptake blocker, RU360 (10 μM), led to an enhancement of the plateau phase of the biphasic Ca2+ response induced by acetylcholine (1 μM). This enhancement was completely abolished by Gd3+; and as has been previously shown for Gd3+, RU360 led to a switch to a wortmannin-sensitive form of exocytosis. Using MitoTracker Red staining we found a close association of mitochondria with the lateral plasma membrane. We propose that in rat pancreatic acinar cells, capacitative Ca2+ entry is targeted directly to mitochondria; and that as a result of Ca2+ uptake, these mitochondria release “third” messengers which both enhance exocytosis and suppress phosphatidylinositol 3-kinase-dependent secretion.