Rates of Na⁺ absorption in the distal nephron increase proportionally with the rates of tubular flow. We tested the hypothesis that the deformation or tension generated in the plasma membrane in response to flow activates the epithelial sodium channel (ENaC). We modified the physical properties of the membrane by changing the temperature and the content of cholesterol. Rates of net Na⁺ absorption measured in cortical collecting ducts (CCDs) perfused at room temperature at slow (∼1) and fast (∼5 nl·min⁻¹·mm⁻¹) flow rates were less than those measured at 37°C at the same flow rates, although increases in tubular fluid flow rates led to comparable relative increases in net Na⁺ absorption at both temperatures. Xenopus laevis oocytes expressing ENaC responded to an increase in shear stress at 22–25°C with a discrete delay followed by a monoexponential increase in whole-cell Na⁺ currents. We observed that temperature affected 1) basal currents, 2) delay times, 3) kinetics of activation, and 4) fold-increase in macroscopic currents in response to flow. The magnitude of the response to flow displayed biphasic behavior as a function of temperature, with a minimal value at 25°C. Steady-state fluorescence anisotropic measurements of purified plasma membranes did not show any obvious phase transition behavior over a temperature range from 8.3°C to 36.5°C. Modification of the content of membrane cholesterol did not affect the response to flow. Our results suggest that the flow-dependent activation of ENaC is not influenced by modifications in the intrinsic properties of the plasma membrane.