The rise in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) is the major trigger for secretion of ACTH hormone (ACTH) from pituitary corticotropes. To better understand the shaping of the Ca²⁺ signal in corticotropes, we investigated the mechanisms regulating the depolarization-triggered Ca²⁺ signal using patch-clamp techniques and indo-1 fluorometry. The rate of cytosolic Ca²⁺ clearance was unaffected by inhibitors of Na⁺/Ca²⁺ exchanger or plasma membrane Ca²⁺-ATPase (PMCA), slightly slowed by sarco-endoplasmic reticulum Ca²⁺-ATPase (SERCA) inhibitor but dramatically slowed by mitochondrial uncouplers, or inhibitor of mitochondrial uniporter. Measurements with rhod-2 revealed that depolarization triggered increase in mitochondrial [Ca²⁺]. Thus, mitochondria have a dominant role in cytosolic Ca²⁺ clearance. Using the Mn²⁺ quench technique, we found the presence of a continuous basal Ca²⁺ influx in corticotropes. This basal Ca²⁺ influx was balanced by the combined actions of mitochondrial uniporter, PMCA and SERCA pumps. Inhibition of the mitochondrial uniporter, PMCA or SERCA pump elevated basal [Ca²⁺]_i. Using membrane capacitance measurement, we found that the change in the shape of the depolarization-triggered Ca²⁺ signal following mitochondrial inhibition was associated with enhancement of the exocytotic response. Thus, mitochondria have a dominant role in the regulation of Ca²⁺ signal and exocytosis in corticotropes.