Restricted expression of activated leukocyte cell adhesion molecule (ALCAM) by hematopoietic cells suggests an important role in the immune system and hematopoiesis. To get insight into the mechanisms that control ALCAM-mediated adhesion we have investigated homotypic ALCAM-ALCAM interactions. Here, we demonstrate that the cytoskeleton regulates ALCAM-mediated cell adhesion because inhibition of actin polymerization by cytochalasin D (CytD) strongly induces homotypic ALCAM-ALCAM interactions. This induction of cell adhesion is likely due to clustering of ALCAM at the cell surface, which is observed after CytD treatment. Single-particle tracking demonstrated that the lateral mobility of ALCAM in the cell membrane is increased 30-fold after CytD treatment. In contrast, both surface distribution and adhesion of a glycosylphosphatidylinositol (GPI)-anchored ALCAM mutant are insensitive to CytD, despite the increase in lateral mobility of GPI-ALCAM upon CytD treatment. This demonstrates that clustering of ALCAM is essential for cell adhesion, whereas enhanced diffusion of ALCAM alone is not sufficient for cluster formation. In addition, upon ligand binding, both free diffusion and the freely dragged distance of wild-type ALCAM, but not of GPI-ALCAM, are reduced over time, suggesting strengthening of the cytoskeleton linkage. From these findings we conclude that activation of ALCAM-mediated adhesion is dynamically regulated through actin cytoskeleton-dependent clustering.