Purinergic signaling is a major pathway in regulating bladder function, and mechanical force stimulates urothelial ATP release, which play an important role in bladder mechanotransduction. Although urothelial ATP release was first reported almost 20 years ago, the way in which release is regulated by mechanical force, and the presence of ATP converting enzymes in regulating the availability of released ATP is still not well understood. Using a set of custom designed Ussing chambers with the ability to manipulate mechanical forces applied on the urothelial tissue, we have demonstrated that it is stretch and not hydrostatic pressure which induces urothelial ATP release. The experiments reveal that urothelial ATP release is tightly controlled by stretch speed, magnitude, and direction. We have further shown that stretch induced urothelial ATP release is insensitive to temperature (4°C). Interestingly, stretch induced ATP release shows polarized distribution, with the ATP concentration in mucosal chamber (nM level) about 10 times higher than the ATP concentration in serosal chamber (sub nM level). Furthermore, we have consistently observed differential ATP lifetime kinetics in the mucosal and serosal chambers, which is consistent with our immunofluorescent localization data showing that ATP converting enzymes ENTPD3 and ALPL are expressed on urothelial basal surface, but not on the apical membrane. In summary, our data indicate that urothelial ATP release is finely regulated by stretch speed, magnitude and direction, and extracellular ATP signaling is likely to be differentially regulated by ectonucleotidase, which results in temporally and spatially distinct ATP kinetics in response to mechanical stretch.