To explore the vascular function of the angiotensin II (ANG II) AT2receptor subtype (AT2R), we generated a vascular smooth muscle cell (SMC) line expressing the AT2R (SMC-vAT2). The involvement of AT2R in the motility response of SMCs was examined in SMC-vAT2 cells and their controls (SMC-v) cultured on either laminin or fibronectin matrix proteins with 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 ANG II significantly inhibited the migration of SMC-vAT2but not SMC-v cells, and this effect was prevented by the AT2R antagonist CGP-42112A. The decreased migration of SMC-vAT2 was not associated with changes in cell growth, cytoskeleton 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 ANG II AT2R inhibits SMC migration via cellular fibronectin synthesis and associated cell binding.