ATP in the renal tubular fluid is an important regulator of salt and water reabsorption via purinergic calcium signaling that involves the P2Y2 receptor, ENaC, and AQP2. Recently, we have shown that connexin (Cx) 30 hemichannels are localized to the non-junctional apical membrane of cells in the distal nephron-collecting duct (CD) and release ATP into the tubular fluid upon mechanical stimuli, leading to reduced salt and water reabsorption. Cx30(-/-) mice show salt-dependent elevations in BP and impaired pressure-natriuresis. Thus, we hypothesized that increased tubular flow rate leads to Cx30-dependent purinergic intracellular calcium ([Ca(2+)]i) signaling in the CD. Cortical CDs (CCDs) from wild type and Cx30(-/-) mice were freshly dissected and microperfused in vitro. Using confocal fluorescence imaging and the calcium-sensitive fluorophore pair Fluo-4 and Fura Red, we found that increasing tubular flow rate from 2 to 20 nl/min caused a significant 2.1-fold elevation in [Ca(2+)]i in wild type CCDs. This response was blunted in Cx30(-/-) CCDs ([Ca(2+)]i increased only 1.2-fold, p < 0.0001 vs. WT, n = 6 each). To further test our hypothesis we performed CD [Ca(2+)]i imaging in intact mouse kidneys in vivo using multiphoton microscopy and micropuncture delivery of the calcium-sensitive fluorophore Rhod-2. We found intrinsic, spontaneous [Ca(2+)]i oscillations in free-flowing CDs of wild type but not Cx30(-/-) mice. The [Ca(2+)]i oscillations were sensitive also to P2-receptor inhibition by suramin. Taken together, these data confirm that mechanosensitive Cx30 hemichannels mediate tubular ATP release and purinergic calcium signaling in the CD which mechanism plays an important role in the regulation of CD salt and water reabsorption.