We propose that fluid shear presents two distinct stimuli to endothelium-the rate of change of flow and flow itself, to which cells sense and respond via independent mechanochemical transduction pathways. We demonstrate that nitric oxide production occurs by two independent mechanisms; a G protein-dependent transient burst stimulated by rapid changes in flow, and a G protein-independent sustained production under steady or smoothly transitioned flow. The novel use of step, ramp, and impulse flow in this study to stimulate nitric oxide production allows the isolation of these individual production events. Impulse flow activates only the G protein-dependent transient burst, which ramp flow fails to stimulate yielding only the sustained response. Step flow, which contains both a rapid increase and a steady flow component, stimulates both pathways, with the response of the superposition of the transient burst and sustained production.