Epithelial cells, both normal and precancerous, stably anchor to basement membranes, whereas malignant tumors pass through them to achieve metastasis. Of basement membrane components, laminin-511 (α5, β1, γ1; LM-511) has been found to be a major isoform in many adult basement membranes. Several studies have shown that LM-511 promotes not only cell adhesion but also tumor cell migration. Thus, LM-511 can be viewed like two distinct molecules in normal vs. tumor cells; tumor cells seem to be able to alter their response (adhesive vs. migratory) to LM-511. In this study we examined the effects of biologically active molecules on A549 lung adenocarcinoma cell adhesion to LM-511. Of them, phorbol 12-myristate 13-acetate (PMA) induced transition to a rounded cell shape and significantly promoted cell migration on LM-511. The attachment of PMA-treated A549 cells to LM-511 was weaker than that of control cells. PMA-stimulated signaling pathway reduced the binding of integrin α3β1 to LM-511. Cell migration assays using inhibitors for signal transduction and cytoskeletal organization showed that suppression of cell adhesion via the rho-associated protein kinase (ROCK) pathway promoted tumor cell migration on LM-511. Our results suggest that the ROCK pathway is involved in the transition from static to migratory cell behaviors on LM-511.