An acute increase in tubular fluid flow rate in the microperfused cortical collecting duct (CCD), associated with a ∼20% increase in tubular diameter, leads to an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i)in both principal and intercalated cells (Woda CB, Leite M Jr, Rohatgi R, and Satlin LM. Am J Physiol Renal Physiol 283: F437-F446, 2002). The apical cilium present in principal but not intercalated cells has been proposed to be a flow sensor. To determine whether flow across the cilium and/or epithelial stretch mediates the [Ca²⁺]_i response, CCDs from New Zealand White rabbits were microperfused in vitro, split-open (to isolate the effect of flow across cilia), or occluded (to examine the effect of stretch and duration/magnitude of the flow impulse), and [Ca²⁺]_i was measured using fura 2. In perfused and occluded CCDs, a rapid (<1 s) but not slow (>3 min) increase in luminal flow rate and/or circumferential stretch led to an approximately threefold increase in [Ca²⁺]_i in both principal and intercalated cells within ∼10 s. This response was mediated by external Ca²⁺ entry and inositol 1,4,5-trisphosphate-mediated release of cell Ca²⁺ stores. In split-open CCDs, an increase in superfusate flow led to an approximately twofold increase in [Ca²⁺]_i in both cell types within ∼30 s. These experimental findings are interpreted using mathematical models to predict the fluid stress on the apical membranes of the CCD and the forces and torques on and deformation of the cilia. We conclude that rapid increases in luminal flow rate and circumferential stretch, leading to shear or hydrodynamic impulses at the cilium or apical membrane, lead to increases in [Ca²⁺]_i in both principal and intercalated cells.