To explore the vascular function of the angiotensin II AT₂ receptor subtype (AT₂R), we generated a vascular smooth muscle cell (SMC) line expressing the AT₂R (SMC-vAT ₂). The involvement of AT₂R in the motility response of SMCs was examined in SMC-vAT ₂ cells and their controls (SMC-v) cultured on either laminin or fibronectin matrix proteins, using the agarose drop technique. All experiments were conducted in the presence of a saturating concentration of losartan to inactivate the AT₁R subtype. Under basal conditions, both cell lines migrated outside drops, but on laminin only. Treatment with angiotensin II significantly inhibited the migration of SMCs-vAT₂ but not of SMCs-v cells, and this effect was prevented by the AT₂R antagonist CGP42112A. The decreased migration of SMCs-vAT₂ was not associated with changes in cell growth, cytoskeletal stiffness, or smooth muscle actin, desmin and tenascin expression. However, it was correlated with increased synthesis and binding of fibronectin. Both responses were prevented by incubation with selective AT₂R antagonists. Addition of GRGDTP peptide, which prevents cell attachment of fibronectin, reversed the AT₂R inhibitory effect on SMC-vAT₂ migration. These results suggest that activated angiotensin II AT₂R inhibits SMC migration via c-FN synthesis and associated cell binding.