The tyrosine kinase Syk is associated with CD18, the β-subunit of the leukocyte adhesion molecules of the β2 integrin family (CD11/CD18), and becomes activated upon β2 integrin-mediated adhesion. In this study, we elucidated the role of Syk in polarization and site-directed migration of neutrophil-like differentiated HL-60 cells and monocytic THP-1 cells. By means of confocal microscopy, we detected a homogenous distribution of Syk in unstimulated cells in suspension. The stimulation of HL-60 cells by formyl-methionyl-leucyl-phenylalanine (fMLP, 100 nM) or the activation of THP-1 cells by monocyte chemoattractant protein-1 (10 ng/ml) induced β2 integrin-mediated cell adhesion and polarization on immobilized fibrinogen which was associated with an enrichment of Syk at the lamellipodium forming site. This effect was abolished by function blocking anti-CD18 antibody or by treatment of the cells with the Syk inhibitor piceatannol (30 μM) suggesting that the redistribution of Syk required both, β2 integrin-mediated adhesion and Syk activation. Moreover, the inhibition of Syk by piceatannol or the downregulation of Syk by antisense technique resulted in an excessive formation of lamellipodia indicating that Syk may act as a negative regulator that limits lamellipodium formation. The analysis of chemotaxis revealed that the inhibition of Syk impaired the ability of the cells to follow a chemotactic gradient whereas random migration was intact. Taken together, our data suggest a novel role for Syk in the maintenance of a bipolar phenotype by regulating lamellipodium formation, which is a critical prerequisite for site-directed migration of leukocytes. © 2005 Wiley-Liss, Inc.