Despite recent advances in our understanding of biochemical regulation of neutrophil chemotaxis, little is known about how mechanical factors control neutrophils’ persistent polarity and rapid motility. Here, by using a human neutrophil-like cell line and human primary neutrophils, we describe a dynamic spatiotemporal pattern of tractions during chemotaxis. Tractions are located at both the leading and the trailing edge of neutrophils, where they oscillate with a defined periodicity. Interestingly, traction oscillations at the leading and the trailing edge are out of phase with the tractions at the front leading those at the back, suggesting a temporal mechanism that coordinates leading edge and trailing edge activities. The magnitude and periodicity of tractions depend upon the activity of non-muscle myosin IIA. Specifically, traction development at the leading edge requires myosin light chain kinase (MLCK)-mediated myosin II contractility and is necessary for 51- integrin activation and leading edge adhesion. Localized myosin II activation induced by spatially activated small GTPase Rho and its downstream kinase p160-ROCK, as previously reported, leads to contraction of actin-myosin II complexes at the trailing edge, causing it to de-adhere. Our data identify a key biomechanical mechanism for persistent cell polarity and motility. ; Post print version of article may differ from published version. The definitive version is available through American Society of Hematology at DOI: 10.1182/blood-2009-12-260851 ; This work was supported in part by NIH grants GM083812 (to F. Wang), GM083601 (to C. Rao and F. Wang), HD059002 (to D. Leckband and F. Wang), GM072744 (to N. Wang), the Illinois Regenerative Medicine Institute (IDPH 2006-05516; to F. Wang), NSF CAREER award 0953267 (to F. Wang) and the Arnold O. Beckman award from the University of Illinois (to F. Wang).