Angiotensin II type 2 (AT ₂ R) or bradykinin B ₂ (B ₂ R) receptor activation enhances NO production. Recently, we demonstrated enhancement of NO production when AT ₂ R and B ₂ R are simultaneously activated in vivo. However, the mechanism involved in this enhancement is unknown. Using confocal fluorescence resonance energy transfer microscopy, we report the distance between the AT ₂ R and B ₂ R in PC12W cell membranes to be 50±5 Å, providing evidence and quantification of receptor heterodimerization as the mechanism for enhancing NO production. The rate of AT ₂ R–B ₂ R heterodimer formation is largely a function of the degree of AT ₂ R–B ₂ R expression. The physical association between the dimerized receptors initiates changes in intracellular phosphoprotein signaling activities leading to phosphorylation of c-Jun terminal kinase, phosphotyrosine phosphatase, inhibitory protein κBα, and activating transcription factor 2; dephosphorylation of p38 and p42/44 mitogen-activated protein kinase and signal transducer inhibitor of transcription 3; and enhancing production of NO and cGMP. Controlling the expression of AT ₂ R–B ₂ R, consequently influencing their biologically active dimerization, presents a potential therapeutic target for the treatment of hypertension and other cardiovascular and renal disorders.