The effects of mitochondrial Ca 2+ uptake on cytosolic Ca 2+ concentration ([Ca 2+] c) were investigated in mouse pancreatic acinar cells using cytosolic and/or mitochondrial Ca 2+ indicators. When calcium stores of the endoplasmic reticulum (ER) were emptied by prolonged incubation with thapsigargin (Tg) and acetylcholine (ACh), small amounts of calcium could be released into the cytosol (Δ[Ca 2+] c=46±6 nM, n=13) by applying mitochondrial inhibitors (combination of rotenone (R) and oligomycin (O)). However, applications of R/O, soon after the peak of Tg/Ach-induced Ca 2+ transient, produced a larger cytosolic calcium elevation (Δ[Ca 2+] c=84±6 nM, n=9), this corresponds to an increase in the total mitochondrial calcium concentration ([Ca 2+] m) by approximately 0.4 mM. In cells pre-treated with R/O or Ru360 (a specific blocker of mitochondrial Ca 2+ uniporter), the decay time-constant of the Tg/ACh-induced Ca 2+ response was prolonged by approximately 40 and 80%, respectively. Tests with the mitochondrial Ca 2+ indicator rhod-2 revealed large increases in [Ca 2+] m in response to Tg/ACh applications; this mitochondrial uptake was blocked by Ru360. In cells pre-treated with Ru360, 10 nM ACh elicited large global increases in [Ca 2+] c, compared to control cells in which ACh-induced Ca 2+ signals were localised in the apical region. We conclude that mitochondria are active elements of cellular Ca 2+ homeostasis in pancreatic acinar cells and directly modulate both local and global calcium signals induced by agonists.