Determining the individual roles of the two dopamine D 1-like receptors (D 1R and D 5R) on sodium transport in the human renal proximal tubule has been complicated by their structural and functional similarity. Here we used a novel D 5R-selective antagonist (LE-PM436) and D 1R or D 5R-specific gene silencing to determine second messenger coupling pathways and heterologous receptor interaction between the two receptors. D 1R and D 5R co-localized in renal proximal tubule cells and physically interact, as determined by co-immunoprecipitation and FRET microscopy. Stimulation of renal proximal tubule cells with fenoldopam (D 1R/D 5R agonist) led to both adenylyl cyclase and phospholipase C (PLC) activation using real-time FRET biosensors ICUE3 and CYPHR, respectively. Fenoldopam increased cAMP accumulation and PLC activity and inhibited both NHE3 and NaKATPase activities. LE-PM436 and D 5R siRNA blocked the fenoldopam-stimulated PLC pathway but not cAMP accumulation, while D 1R siRNA blocked both fenoldopam-stimulated cAMP accumulation and PLC signaling. Either D 1R or D 5R siRNA, or LE-PM436 blocked the fenoldopam dependent inhibition of sodium transport. Further studies using the cAMP-selective D 1R/D 5R agonist SKF83822 and PLC-selective D 1R/D 5R agonist SKF83959 confirmed the cooperative influence of the two pathways on sodium transport. Thus, D 1R and D 5R interact in the inhibition of NHE3 and NaKATPase activity, the D 1R primarily by cAMP, while the D 1R/D 5R heteromer modulates the D 1R effect through a PLC pathway.