This study investigates ionic mechanisms involved in growth arrest induced by extracellular ATP in androgen-independent prostate cancer cells. Extracellular ATP reversibly induced a rapid and sustained pHi decrease from 7.41 to 7.11. Inhibition of calcium influx, lowering extracellular calcium and buffering cytoplasmic calcium inhibited ATP induced acidification, thereby demonstrating that acidification is a consequence of calcium entry. We show that ATP induced reuptake of calcium by the mitochondria and a transient depolarization of the inner mitochondrial membrane. ATP-induced acidification was reduced following the dissipation of the mitochondrial proton gradient by rotenone and FCCP, following inhibition of calcium uptake into the mitochondria by ruthenium red and following inhibition of the F0F1 ATPase with oligomycin. ATP induced acidification was not induced by a stimulation of the Cl-/HCO3- exchanger nor by an inhibition of the Na+/H+ exchanger. In addition, intracellular acidification, induced by an ammonium prepulse method, reduced the amount of releasable calcium from the endoplasmic reticulum assessed by measuring change in cytosolic calcium induced by thapsigargin or by ATP in a calcium free medium. This later finding reveals a cross talk between pHi and calcium homeostasis where the calcium induced-intracellular acidification can in turn regulates the amount of calcium that can be released from endoplasmic reticulum. Furthermore pHi decrease was capable of reducing cell growth. Taken together our result let us to suggest that ATP induced acidification in DU-145 cells results from specific effect off the mitochondrial function and is one of the major mechanisms leading to growth arrest induced by ATP.