Agonist-evoked cytosolic Ca 2+ spikes in mouse pancreatic acinar cells are specifically initiated in the apical secretory pole and are mostly confined to this region. The role played by mitochondria in this process has been investigated. Using the mitochondria-specific fluorescent dyes MitoTracker Green and Rhodamine 123, these organelles appeared as a bright belt concentrated mainly around the secretory granule area. We tested the effects of two different types of mitochondrial inhibitor on the cytosolic Ca 2+ concentration using simultaneous imaging of Ca 2+-sensitive fluorescence (Fura 2) and electrophysiology. When carbonyl cyanide m-chlorophenylhydrazone (CCCP) was applied in the presence of the Ca 2+-releasing messenger inositol 1, 4, 5-trisphosphate (IP 3), the local repetitive Ca 2+ responses in the granule area were transformed into a global rise in the cellular Ca 2+ concentration. In the absence of IP 3, CCCP had no effect on the cytosolic Ca 2+ levels. Antimycin and antimycin+ oligomycin had the same effect as CCCP. Active mitochondria, strategically placed around the secretory pole, block Ca 2+ diffusion from the primary Ca 2+ release sites in the granule-rich area in the apical pole to the basal part of the cell containing the nucleus. When mitochondrial function is inhibited, this barrier disappears and the Ca 2+ signals spread all over the cytosol.