We recently showed that the motility of the malignant Calu-1 human epidermoid lung carcinoma cells correlates to their expression levels of α2, α3, α6, and β1 integrin subunits. To determine a causative relationship underlying this correlation, here we measured Calu-1 cell adhesion to and migration on laminin, collagen IV, human umbilical vein endothelial cell monolayers, and endothelial cell extracellular matrix in the presence of function-blocking antibodies against the suspect integrin subunits. Blocking individual α subunits did not affect adhesion to or motility on laminin, but when used in pair-wise combinations, monoclonal antibody treatments significantly decreased tumor cell motility on, without diminishing adhesion to, laminin and the other substrates. Blocking all three α subunits at once or the β1 subunit alone abolished migration on laminin; however, the latter treatment also abolished adhesion, whereas the former treatment did not. By contrast, blocking the β1 subunit significantly reduced motility on collagen IV, endothelial cell monolayers, and endothelial cell extracellular matrix, but always without affecting adhesion. These results suggest a separation of roles and mechanisms of different integrins in adhesion and motility.