Collecting duct-derived endothelin (ET)-1 is an autocrine inhibitor of Na+ and water reabsorption; its deficiency causes hypertension and water retention. Extracellular fluid volume expansion increases collecting duct ET-1, thereby promoting natriuresis and diuresis; however, how this coupling between volume expansion and collecting duct ET-1 occurs is incompletely understood. One possibility is that volume expansion increases tubular fluid flow. To investigate this, cultured IMCD3 cells were subjected to static or flow conditions. Exposure to a shear stress of 2 dyn/cm2 for 2 h increased ET-1 mRNA content by ~2.3-fold. Absence of perfusate Ca2+, chelation of intracellular Ca2+, or inhibition of Ca2+ signaling (calmodulin, Ca2+/calmodulin-dependent kinase, calcineurin, PKC, or phospholipase C) prevented the flow response. Evaluation of possible flow-activated Ca2+ entry pathways revealed no role for transient receptor potential (TRP)C3, TRPC6, and TRPV4; however, cells with TRPP2 (polycystin-2) knockdown had no ET-1 flow response. Flow increased intracellular Ca2+ was blunted in TRPP2 knockdown cells. Nonspecific blockade of P2 receptors, as well as specific inhibition of P2X7 and P2Y2 receptors, prevented the ET-1 flow response. The ET-1 flow response was not affected by inhibition of either epithelial Na+ channels or the mitochondrial Na+/Ca2+ exchanger. Taken together, these findings provide evidence that in IMCD3 cells, flow, via polycystin-2 and P2 receptors, engages Ca2+- dependent signaling pathways that stimulate ET-1 synthesis.