We have generated a monoclonal antibody (MAb) L1A3 directed to the alpha v integrin subunit as shown by competitive binding with other anti-alpha v-specific MAbs and immunodepletion. MAb L1A3 is a function-blocking antibody inhibiting cell adhesion to the extracellular matrix proteins, fibronectin and vitronectin. Adherence to vitronectin of all cells studied including normal dermal microvascular endothelial cells and three tumor cell lines was inhibited in the presence of MAb L1A3. However, the contribution of the alpha v integrin subunit in mediating adhesion to fibronectin was dependent on the cell line, as indicated by differences in the inhibition of cell adhesion with MAb L1A3 and alpha 5 beta 1 integrin subunit blocking MAb P1D6. Glioma U251.3 cell adhesion to fibronectin was blocked by either MAb L1A3 or MAb P1D6 while fibrosarcoma HT1080 cells were blocked with MAb P1D6 only. Tumor cell migration mediated by vitronectin and fibronectin is blocked by MAb L1A3 in the two-dimensional spheroid outgrowth assay. Microvascular endothelial cell transwell membrane migration onto the fibronectin was also blocked by MAb L1A3. Comparison of the integrins involved in U251.3 cell migration on fibronectin or tenascin using a panel of integrin blocking MAbs including MAb L1A3 showed that only a subset of integrins participating in cell adhesion is essential for cell migration and these integrins appear to be ligand specific. Fibronectin-mediated tumor cell migration was critically dependent on alpha v integrins as shown by L1A3 blocking of migration while the beta 1 integrins were absolutely necessary for tenascin-mediated cell migration.