Shear stress differentially activates extracellular signal-regulated kinase (ERK) and c-Jun NH₂-terminal kinase (JNK) by mechanisms involving Gα_i2 and Gβ/γ proteins, respectively, in bovine aortic endothelial cells (BAEC). The early events in this signaling mechanism by which G proteins regulate ERK and JNK in response to shear stress have not been defined. Here we show that BAEC endogenously express a G protein-dependent form of phosphatidylinositol 3-kinase, PI3Kγ, and its activity is stimulated by shear stress. PI3Kγ activity was measured in vitro using BAEC that were transiently transfected with an epitope-tagged PI3Kγ (vsv-PI3Kγ). Exposure of BAEC to shear stress rapidly and transiently stimulated the activity of vsv-PI3Kγ (maximum by 15 s, with a return to basal after 1-min exposure to 5 dyn/cm² shear stress). Activity of vsv-PI3Kγ was stimulated by shear stress intensities as low as 0.5 dyn/cm². Treatment of BAEC with an inhibitor of PI3K, wortmannin, inhibited shear-dependent activation of JNK but had no effect on that of ERK. Furthermore, expression of a kinase-inactive mutant (PI3Kγ^K799R) in BAEC inhibited the shear-dependent activation of JNK but not ERK. Taken together, these results suggest that PI3Kγ selectively regulates the shear-sensitive JNK pathway. This differential and novel signaling pathway may be responsible for coordinating various mechanosensitive events in endothelial cells.