Integrins are highly regulated receptors that can function in both cell–substrate and cell–cell adhesion. We have found that the activating anti-β1 mAb, 12G10, can specifically and rapidly induce both cell–substrate and cell–cell adhesion of HT-1080 human fibrosarcoma and other cell types. Binding of mAb 12G10 induced clustering of cell–surface integrins, and the preferential localization of β1 integrins expressing the 12G10 epitope at cell–cell adhesion sites. Fab fragments of mAb 12G10 induced HT-1080 cell–cell adhesion as effectively as did intact antibodies, suggesting that integrin clustering was not due to direct antibody crosslinking. Latrunculin B, an inhibitor of F-actin polymerization, inhibited cell–cell adhesion but not the clustering of integrins. Results from a novel, two-color cell–cell adhesion assay suggested that nonactivated cells can bind to activated cells and that integrin activation-induced HT-1080 cell–cell adhesion minimally requires the interaction of activated α2β1 with nonactivated α3β1. These findings suggest that HT-1080 cell–cell adhesion induced by integrin activation require a signaling process involving integrin clustering and the subsequent organization of the cytoskeleton. Integrin activation could therefore play a key role in cell–cell adhesion.