Leukocyte migration requires cell polarization which is characterized by the formation of a lamellipodium and an enrichment of specific signaling components at the leading edge. The non-receptor tyrosine kinase Syk interacts with the phosphoinositide 3-kinase (PI3K) which is essential for leukocyte polarization and migration. To address the question whether Syk-mediated signaling via PI3K is required for leukocyte polarization and migration, we generated neutrophil-like differentiated HL-60 (dHL-60) cells transiently expressing wildtype Syk (EGFP-Syk) or a Syk mutant lacking the binding site for PI3K (EGFP-Syk Y323F) tagged with an enhanced green fluorescent protein. By means of fluorescence microscopy, we found that EGFP-Syk as well as EGFP-Syk Y323F was enriched at the lamellipodium of the transfected dHL60 cells upon formyl-petide-induced cell polarization. However, the expression of EGFP-Syk Y323F induced the formation of multiple lamellipodia whereas the majority of the EGFP-Syk control cells polarized properly and formed one lamellipodium. Moreover, time-lapse video microscopy revealed that the Syk Y323F transfectants were characterized by a spatial and temporal instability of the leading edge. Taken together, our data suggest that Syk-mediated PI3K signaling is involved in the stabilization of cell polarity which represents an important prerequisite for efficient leukocyte migration. Supported by DFG (WA 1048/2-1).