Vascular endothelial cells respond to extracellular ATP by InsP₃-mediated Ca²⁺ release from the endoplasmic reticulum (ER) followed by Ca²⁺ influx and subsequent synthesis of vasodilators. In this study, the contribution of mitochondria in shaping the ATP-induced Ca²⁺ increase was examined in ovine uterine artery endothelial cells from nonpregnant and pregnant (late gestation) ewes (NP- and P-UAEC, passage 4). The mitochondrial protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) induced a rapid mitochondrial depolarization. CCCP also slowly increased [Ca²⁺]_c, which then gradually declined to 10-20 nM above resting level. Pretreatment with CCCP for 30 min significantly inhibited both ATP and thapsigargin-induced [Ca²⁺]_c, with NP-UAEC > P-UAEC. Pretreatment of mitochondrial permeability transition pore (MPTP) inhibitor cyclosporine A (CsA) did not affect CCCP-induced mitochondrial depolarization, but delayed CCCP-induced [Ca²⁺]_c for about 12-15 min (we termed this the "window of time"). During the CsA-delayed "window of time" of CCCP-induced [Ca²⁺]_c, ATP induced a normal Ca²⁺ response, but after this "window of time", ATP-induced [Ca²⁺]_c was significantly inhibited. Pretreatment of oligomycin B to prevent intracellular ATP depletion by F0F1-ATPase, did not reduce the inhibition of ATP-induced [Ca²⁺]_c by CCCP. Ruthenium red, a mitochondrial Ca²⁺ uptake blocker, did not mimic the inhibition of Ca²⁺ signaling by CCCP. In conclusion, our data show that mitochondrial Ca²⁺ depletion following dissipation of mitochondrial membrane potential () with CCCP inhibits ATP-induced [Ca²⁺]_c, mediated at the level Ca²⁺ release from the ER. Moreover, our data revealed that P-UAEC is more resistant to inhibitory effect of CCCP on [Ca²⁺]_c than NP-UAEC.