We have characterized the effect of the Ca²⁺-ATPase inhibitors 2,5-di-(t-butyl)-1,4-benzohydroquinone (tBHQ) and thapsigargin on the concentration of cytosolic Ca²⁺ in single bovine adrenal chromaffin cells by video-imaging of fura-2-loaded cells. Addition of either inhibitor released Ca²⁺ from internal stores in the absence of external Ca²⁺ tBHQ was unable to stimulate further Ca²⁺ release after addition of thapsigargin, but thapsigargin could do so after release by tBHQ, indicating that the tBHQ-sensitive stores are a sub-set of those sensitive to thapsigargin. Angiotensin II was able to elicit Ca²⁺ release after application of tBHQ, indicating that at least part of the tBHQ-sensitive stores were distinct from those discharged by Ins(1,4,5)P₃. In the presence of external Ca²⁺, both Ca²⁺-ATPase inhibitors produced a more prolonged rise in cytosolic Ca²⁺ consistent with stimulated Ca²⁺ entry. The ability of the inhibitors to activate a Ca²⁺-entry pathway was confirmed by monitoring quenching of fura-2 after stimulated entry of the Ca²⁺ surrogate Mn²⁺. These findings indicate that bovine adrenal chromaffin cells possess a mechanism by which Ca²⁺ entry can be activated. following emptying of certain internal stores, independently of receptor occupation.