The vascactive peptide urotensin-II (hU-II) has been associated with vascular remodelling processes in pulmonary hypertension (PH). hU-II acts via the G-protein coupled receptor GPR14. However, the mechanisms mediating GPR14-signalling remain unclear. ROS generated by NADPH oxidases can act as signalling molecules in vascular cells. Since the GTPase Rac-1 is required for activation of this enzyme we investigated whether hU-II and GPR14 can activate Rac-1 and ROS production in pulmonary artery smooth muscle cells (PASMC). hU-II rapidly activated Rac-1 and this response was abolished by the GPR14 antagonist urantide. hU-II also increased ROS levels within 5 minutes. Overexpression of GPR14 enhanced ROS production by hU-II, whereas urantide, dominant-negative RacT17N or pertussis toxin inhibited hU-II-induced Rac-1 activation and ROS generation indicating coupling of GPR14 to Gai proteins. Indeed, transfection of dominant-negative Gai3 completely diminished hU-II-stimulated ROS generation and Rac-1 activation. These results show that hU-II is a potent activator of Rac-1 leading to rapid ROS generation involving GPR14 and Gai proteins. hU-II- mediated activation of the NADPH oxidase in PASMC may thus contribute to vascular remodeling.