AbstractThe secretory granules constitute one of the less well‐known compartments in terms of Ca2+ dynamics. They contain large amounts of total Ca2+, but the free intragranular [Ca2+] ([Ca2+]SG), the mechanisms for Ca2+ uptake and release from the granules and their physiological significance regarding exocytosis are still matters of debate. We used in the present work an aequorin chimera targeted to the granules to investigate [Ca2+]SG homeostasis in bovine adrenal chromaffin cells. We found that most of the intracellular aequorin chimera is present in a compartment with 50–100 μm Ca2+. Ca2+ accumulation into this compartment takes place mainly through an ATP‐dependent mechanism, namely, a thapsigargin‐sensitive Ca2+‐ATPase. In addition, fast Ca2+ release was observed in permeabilized cells after addition of inositol 1,4,5‐trisphosphate (InsP3) or caffeine, suggesting the presence of InsP3 and ryanodine receptors in the vesicular membrane. Stimulation of intact cells with the InsP3‐producing agonist histamine or with caffeine also induced Ca2+ release from the vesicles, whereas acetylcholine or high‐[K+] depolarization induced biphasic changes in vesicular [Ca2+], suggesting heterogeneous responses of different vesicle populations, some of them releasing and some taking up Ca2+ during stimulation. In conclusion, our data show that chromaffin cell secretory granules have the machinery required for rapid uptake and release of Ca2+, and this strongly supports the hypothesis that granular Ca2+ may contribute to its own secretion.