Endothelium-dependent vasoactive substances are known to evoke complex changes in the endothelial membrane potential (MP) and to increase intracellular pH in endothelial cells (EC). In our present study, we investigated the effect of agents able to increase intracellular pH on the MP of intact guinea pig aortic EC, and also the effect of blocking of Na+-H+ exchanger on ATP-induced electrical responses. Intracellular alkalinization was induced either by addition of ammonium chloride (NH4Cl) to the superfusate, or by changing the bath solution saturated with 10% CO2+90% O2 to a solution saturated with 100% O2. Both approaches evoked hyperpolarization of EC. After intracellular Ca2+ chelation by pretreatment of aortic preparations with 20 μM BAPTA-AM, the amplitude of NH4Cl-induced hyperpolarization dropped from 3.9±0.6 to 0.7±0.3 mV. After pretreatment with ATP, NH4Cl-induced hyperpolarization was not abolished, whereas after caffeine pretreatment this hyperpolarization was not observed. In the Na+-free solution and in the presence of furosemide, ATP-evoked hyperpolarization became longer. The same effect was also observed in the presence of sodium acetate, which directly acidifies the cytosol. In the Ca2+-free solution, furosemide did not induce prolongation of ATP-evoked hyperpolarization. Taking into account the results, it could be proposed that, first, hyperpolarization of EC after intracellular alkalinization is a result of Ca2+ release from the intracellular stores sensitive both to an increase in intracellular pH and to caffeine application. Second, intracellular alkalinization, being a result of activation of Na+-H+-antiporter, inhibits influx of extracellular Ca2+ into EC under ATP stimulation.