Endothelial cells release nitric oxide (NO) more potently in response to increased shear stress than to agonists which elevate intracellular free calcium concentration ([Ca²⁺]_i). To determine mechanistic differences in the regulation of endothelial constitutive NO synthase (ecNOS), we measured NO production by bovine aortic endothelial cells exposed to shear stress in a laminar flow chamber or treated with Ca²⁺ ionophores in static culture. The kinetics of cumulative NO production varied strikingly: shear stress (25 dyne/cm²) stimulated a biphasic increase over control that was 13-fold at 60 minutes, whereas raising [Ca²⁺]_i caused a monophasic 6-fold increase. We hypothesized that activation of a protein kinase cascade mediates the early phase of flow-dependent NO production. Immunoprecipitation of ecNOS showed a 210% increase in phosphorylation 1 minute after flow initiation, whereas there was no significant increase after Ca²⁺ ionophore treatment. Although ecNOS was not tyrosine-phosphorylated, the early phase of flow-dependent NO production was blocked by genistein, an inhibitor of tyrosine kinases. To determine the Ca²⁺ requirement for flow-dependent NO production, we measured [Ca²⁺]_i with a novel flow-step protocol. [Ca²⁺]_i increased with the onset of shear stress, but not after a step increase. However, the step increase in shear stress was associated with a potent biphasic increase in NO production rate and ecNOS phosphorylation. These studies demonstrate that shear stress can increase NO production in the absence of increased [Ca²⁺]_i, and they suggest that phosphorylation of ecNOS may importantly modulate its activity during the imposition of increased shear stress.