EKODE, an epoxy-keto derivative of linoleic acid was previously shown to stimulate aldosterone secretion in rat adrenal glomerulosa cells (15). In the present study, we investigated the effect of exogenous EKODE on cytosolic [Ca²⁺] increase and aimed to elucidate the mechanism involved in this process. Through the use of fluorescent Ca²⁺ -sensitive dye Fluo-4, EKODE was shown to rapidly increase [Ca²⁺]_i along a bell-shaped dose-response relationship with a maximum peak at 5 µM. Experiments performed in the presence or absence of Ca²⁺ revealed that this increase in [Ca²⁺]_i originated exclusively from intracellular pools. EKODE-induced [Ca²⁺]_i increase was blunted by prior application of Angiotensin II, Xestospongin C and CPA indicating that InsP₃-sensitive Ca²⁺ stores can be mobilized by EKODE in spite of the absence of InsP₃ production. Accordingly, EKODE response was not sensitive to the PLC inhibitor U-73122. EKODE mobilized a Ca²⁺ store included in the thapsigargin-sensitive stores although the interaction between EKODE and TG appears complex since EKODE added at the plateau response of TG induced a rapid drop in [Ca²⁺]_i. 9-oxo-octadecadienoic acid, another oxidized derivative of linoleic acid, also increases [Ca²⁺]_I with a dose-response curve similar to EKODE. However, arachidonic and linoleic acids at 10 µM failed to increase [Ca²⁺]_i but did reduce the amplitude of the response to EKODE. It is concluded that EKODE mobilizes Ca²⁺ from an InsP₃-sensitive store and that this [Ca²⁺]_i increase is responsible for aldosterone secretion by glomerulosa cells. Similar bell-shaped dose-response curves for aldosterone and [Ca²⁺]_i increase reinforce this hypothesis.