To address pivotal roles of cell surface F₁/F_O ATP synthase in the development of acidic microenvironment in tumor tissues, we investigated effects of shear stress stimulation on the cultured human breast cancer cells, MDA-MB-231 and MDA-MB-157 or human melanoma cells, SK-Mel-1. Shear stress stimulation (0.5-5.0 dyn/cm2), the levels of which are similar to those produced by the interstitial flow, induced strength-dependent co-release of ATP and H⁺ from the cells, which triggered CO₂ gas excretion. In contrast, the same level of shear stress stimulation did not induce significant ATP release and CO₂ gas excretion from the control human mammary epithelial cells (HMEC). Marked immunocytochemical and mRNA expression of cell surface F₁/F_O ATP synthase, vacuolar-ATPase (V-ATPase), carbonic anhydrase type IX, and ectonucleoside triphosphate diphosphohydrolase (ENTPDase) 3 were detected in MDA-MB-231 cells, but little or no expression on the HMEC. Pretreatment with cell surface F₁/F_O ATP synthase inhibitors, but not cell surface V-ATPase inhibitors caused a significant reduction of the shear stress stimulation-mediated ATP release and CO₂ gas excretion from MDA-MB-231 cells. The ENTPDase activity in the shear stress-loaded MDA-MB-231 cell culture medium supernatant increased significantly in a time-dependent manner. In addition, MDA-MB-231 cells displayed strong staining for purinergic 2Y1 (P2Y1) receptors on their surfaces, and the receptors partially co-localized with ENTPDase 3. These findings suggest that cell surface F₁/F_O ATP synthase, but not V-ATPase may play key roles in the development of interstitial flow-mediated acidic microenvironment in tumor tissues through the shear stress stimulation-induced ATP and H⁺ co-release and CO2 gas production.