Anchorage-independent myelomonocytic cells acquire adherence within minutes of differentiation stimuli, such as the proteolytically inactive N-terminal fragment of urokinase binding to its cognate glycosylphosphatidylinositol (GPI)-anchored receptor. Here, we report that urokinase-treated differentiating U937 monocyte-like cells exhibit a rapid and transient inhibition of p56/59hck and p55fgr whereas no changes in the activity of other Src family kinases, such as p53/56lyn and p59fyn were observed. U937 transfectants expressing a kinase-defective (Lys267 to Met) p56/59hck variant exhibit enhanced adhesiveness and a marked F-actin redistribution in thin protruding structures. Conversely, urokinase as well as expression of wild-type or constitutively active (Tyr499 to Phe) p56/59hck stimulates the directional migration of uninduced U937 cells. Accordingly, expression of constitutively active or kinase inactive p56/59hck selectively prevents urokinase receptor-dependent induction of either adhesion or motility, indicating that a specific activation state of p56/59hck is required for each cell response. In conclusion, modulation of the intracellular p56/59hck tyrosine kinase activity switches cell motility towards adherence, providing a mutually exclusive mechanism to regulate these properties during monocyte/macrophage differentiation in vivo.