We previously found that adenosine stimulates ATP release from Madin-Darby canine kidney (MDCK) cells, by activating an Ins(1,4,5)P(3) sensitive-calcium (Ca(2+)) pathway through the stimulation of A(1) receptors. Thus, we investigated the intracellular pathway of ATP efflux after the rise in intracellular Ca(2+) in MDCK cells. Adenosine evoked an increase in mitochondrial Ca(2+) using Rhod-2/AM, a mitochondrial Ca(2+) indicator. Adenosine-induced ATP release was inhibited by mitochondrial modulators, such as oxidative phosphorylation modulators (carbonyl cyanide 3-chlorophenylhydrazone and oligomycin), mitochondrial ADP/ATP carrier inhibitors (N-ethylmaleimide, carboxyatractyloside and bongkrekic acid), a mitochondrial Na(+)-Ca(2+) exchange inhibitor (CGP-37157). In addition, mitochondrial modulators significantly reduced intracellular ATP content. On the other hand, 2-deoxy-glucose (2-DG) induced a greater decrease in intracellular ATP content than mitochondrial modulators. ATP release was still induced by adenosine in the presence of 5mM 2-DG. These results suggest that mitochondria play an important role in the signaling pathway of adenosine-triggered ATP release in MDCK cells.