Extracellular matrix receptors on ductus arteriosus smooth muscle cells (SMC) must enable the cells to migrate through both interstitial and basement membrane matrices to form intimal mounds during postnatal ductus closure. We examined the role of β₁ and β₃ integrin receptors on SMC adhesion and migration. Using a new assay to measure cell migration, we found that lamb ductus arteriosus SMC attach to and migrate over surfaces coated with fibronectin (FN), laminin (LN), vitronectin (VN), and collagens I (I) and IV (IV). Blocking antibodies, specific to different integrin complexes, showed that SMC adhesion to FN, LN, I, and IV depended exclusively on functioning β₁ integrins with little, if any, contribution by the α_vβ₃ integrin; on the other hand, cell migration over these substrates depended to a large extent on the α_vβ₃ receptor. Immunofluorescent staining demonstrated that during the early phase of SMC migration, the β₁ integrins organized rapidly into focal plaques that, with time, gradually covered the cell's basal surface; on the other hand, the β₃ receptor remained concentrated at all times at the cell's margins. Ligand affinity chromatography and immunoprecipitation techniques identified a unique series of β₁ integrins binding to each matrix component: FN (α₅β₁, α₃β₁, α_vβ₁), LN (α₁β₁, α₇β₁), VN (α_vβ₁), I (α₁β₁, α₂β₁), and IV (α₁β₁). In contrast, the β₃ integrin, α_vβ₃, bound to all the substrates tested: FN, LN, VN, I, and IV. The results indicate that β₁ and β₃ integrins may play different roles in attachment and migration as SMC move through the vascular extracellular matrix to produce obliteration of the ductus arteriosus lumen.