The homeostatic control of blood pressure hinges upon the delicate balance between pro-hypertensinogenic (e.g., sympathetic nervous system) and anti-hypertensinogenic systems (e.g., dopamine system). The D1-like dopamine receptors (D1R and D5R) and the α1A adrenergic receptor (ARα1A) are endogenously expressed in the renal proximal tubules and engender opposing effects on sodium transport, i.e., natriuresis (D1R and D5R) or anti-natriuresis (ARα1A). We tested the hypothesis that the D1R and D5R interact with and regulate the ARα1A in human renal proximal tubule cells (hRPTCs) and in mice. We found that the D1R and D5R colocalized with the ARα1A in hRPTCs and in proximal tubules in human kidney sections. Both receptors immunoprecipitated, pulled-down, and co-fractionated with ARα1A in lipid rafts. Short-term co-treatment with fenoldopam (1 μM, 15 min) reversed the ARα1 agonist phenylephrine (10 μM, 15 min)-induced Na+,K+-ATPase (NKA) translocation from the cytosol to the plasma membrane in hRPTCs (plasma membrane NKA: vehicle=100±5% vs. fenoldopam=65±3% vs. phenylephrine=177±5% vs. co-treatment=115±7%; P<0.05, n=3-4). Long-term fenoldopam (1 μM, 24 hr) treatment resulted in decreased D1R (70.0±5.9%, P<0.05, n=3) and D5R (50.1±10.7%, P<0.05, n=3), consistent with D1-like receptor desensitization, but increased ARα1A abundance (142.6±4.3%, P<0.05, n=3) in hRPTCs. RNAi silencing of ARα1A (48 hr) increased the expression of D1R and D5R. To determine the extent of regulation of each D1-like receptor on ARα1A, we used the subclass-selective ARα1 agonist phenylephrine (5 μg/kg body weight, i.p.) and the receptor-specific ARα1A agonist A610603 (25 ng/kg body weight, i.p.) to inhibit Na+ excretion in three mouse strains. Phenylephrine treatment resulted in 59.6%, 84.2%, 99.3%, and 99.5% reduction from basal level of 24-hr Na+ excretion_while A61603 treatment resulted in 42.4%, 67.1%, 99.9%, and 100% reduction_in wild-type controls, D1R-/-, and D5R-/- knockout mice, and D1R/D5R-/- double knockout mice, respectively, suggesting a stronger regulatory effect of D5R on ARα1A. Elucidating the intricacies of the interaction among these receptors is crucial for a better understanding of the crosstalk between anti- and pro-hypertensive systems.