D5 dopamine receptor (D5R) knock-out mice (D5(-/-)) have a higher blood pressure (BP) and higher reactive oxygen species (ROS) production than their D5R wild-type littermates (D5(+/+)). We tested the hypothesis that the high BP and increased ROS production in D5(-/-) mice may be caused by decreased heme oxygenase-1 (HO-1) expression and activity. We found that renal HO-1 protein expression and HO enzyme activity were decreased (65 and 50%, respectively) in D5(-/-) relative to D5(+/+) mice. A 24 h of administration of hemin, an HO-1 inducer, increased HO-1 expression and HO activity (6.8- and 1.9-fold, respectively) and normalized the increased ROS production and BP in D5(-/-) mice. Expression of HO-1 protein and HO activity were increased (2.3- and 1.5-fold, respectively) in HEK cells that heterologously expressed human wild-type D5R (HEK-hD5R), but not the empty vector-transfected HEK-293 cells. Fenoldopam (Fen), a D5R agonist, increased HO activity (3 h), HO-1 protein expression, HO-1 and D5R colocalization and co-immunoprecipitation in HEK-hD5R cells. Cellular NADPH oxidase activity was decreased by 35% in HEK-hD5R that was abrogated with silencing of the heme oxygenase 1 gene (HMOX1). HMOX1 siRNA also impaired the ability of Fen to decrease NADPH oxidase activity in HEK-hD5R cells. In summary, the D5R positively regulates HO-1 through direct protein/protein interaction in the short-term and by increasing HO-1 protein expression in the long-term. The impaired D5R regulation of HO-1 and ROS production contributes to the pathogenesis of hypertension in D5(-/-) mice.