We have shown that leukocytes retract their pseudopods and detach from substrates after exposure to physiological fluid shear stresses (≈1.5 dyn/cm2). In inflammation, however, pseudopod projection during spreading and firm adhesion on endothelium is observed even in microvessels with normal blood flow and fluid shear stresses. Thus, we examined mechanisms that may serve to regulate the shear stress response of circulating leukocytes. In the presence of inflammatory mediators (platelet-activating factor [PAF] f-met-leu-phe), a subgroup of cells ceases to respond to shear stress. cGMP analogs and nitric oxide (NO) donors enhance the shear stress response and reverse the inhibitory effect of inflammatory mediators on the shear stress response, whereas depletion of cGMP leads to cessation of the shear stress response even in unstimulated leukocytes. The ability of cGMP to enhance the shear stress response is not associated with CD18 expression, because cGMP has no effect on CD18 expression in response to shear stress. The shear stress response of leukocytes in endothelial nitric oxide synthase (−/−) mice, in which NO level in blood is decreased, is attenuated compared with that in wild-type mice. In rat mesentery venules stimulated by PAF under normal blood flow, a cGMP analog diminishes pseudopod projection of leukocytes, whereas inhibition of NO leads to enhanced pseudopod projection and spreading. The evidence suggests that inflammatory mediators suppress the shear stress response of leukocytes leading to spreading even under normal physiological shear stress, whereas cGMP may serve to maintain shear stress response even in inflammation. The full text of this article is available at http://www.circresaha.org.