The requirement for phosphatidylinositol 3-kinase (PI3K) in the establishment of cell polarity and motility in a number of cell types has recently come into question. In this study, we demonstrate that inhibition of PI3K by wortmannin in neutrophil-like differentiated HL60 cells expressing CXCR2 resulted in reduced cell motility but normal chemotaxis in response to a gradient of CXCL8. However, wortmannin inhibition of PI3K did impair the ability of cells to re-orient their polarity and respond quickly to a change in the direction of the CXCL8 gradient. We hypothesized that Src-regulated ELMO-Dock2-Rac2 activation mediates chemotaxis in the absence of PI3K activity. Inhibition of Src with the small molecule inhibitor, PP2, or inhibition of Dock2 by shRNA knockdown confirmed the functional role of Src and Dock2 in regulating chemotaxis when PI3K was inhibited. Moreover, neutrophils isolated from bone marrow of hck(-/-)fgr(-/-)lyn(-/-) mice exhibited much more severe inhibition of chemotaxis when PI3K was blocked with wortmannin as compared with neutrophils isolated from bone marrow of wild-type mice. Thus, PI3K and Src-ELMO-Dock2 pathways work in parallel to activate Rac2 and modulate chemotaxis in response to a CXCL8 gradient in neutrophils.