AlF₄^- is known to generate [Ca²⁺]_i oscillations by activating G-proteins in many cell types. However, in rat pancreatic acinar cells, AlF₄^--evoked [Ca²⁺]_i oscillations were reported to be dependent on extracellular Ca²⁺, which contrasts with the [Ca²⁺]_i oscillations induced by cholecystokinin (CCK). Therefore, we investigated the mechanisms by which AlF₄^- generates extracellular Ca²⁺-dependent [Ca²⁺]_i oscillations in rat pancreatic acinar cells. AlF₄^--induced [Ca²⁺]_i oscillations were stopped rapidly by the removal of extracellular Ca²⁺ and were abolished upon the addition of 20 mM caffeine and 2 µM thapsigargin, indicating that Ca²⁺ influx plays a crucial role in the maintenance of the oscillations and that an IP₃-sensitive Ca²⁺ store is also required. The amount of Ca²⁺ in the intracellular Ca²⁺ store was found to decrease as the AlF₄^--induced [Ca²⁺]_i oscillations continued. Measurement of ⁴⁵Ca²⁺ influx into isolated microsomes revealed that AlF₄^- directly inhibited sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA). The activity of plasma membrane Ca²⁺ ATPase (PMCA) during AlF₄^- stimulation was not significantly different from that during CCK stimulation. After the partial inhibition of SERCA with 1 nM thapsigargin, 20 pM CCK-evoked [Ca²⁺]_i oscillations were found to be dependent on extracellular Ca²⁺. This study shows that AlF₄^- induces [Ca²⁺]_i oscillations probably by IP₃ production via G-protein activation but that these oscillations are strongly dependent on extracellular Ca²⁺ due to the partial inhibition of SERCA.