Angiotensin (Ang) AT2 receptors (AT2Rs) are expressed in the kidney at vascular and tubular sites. Previous studies from our lab have shown that renal AT2R activation induces natriuresis and that Ang III is the endogenous agonist for this response. Compound 21 (C21) is a highly selective non-peptide AT2R agonist with approx. 25,000-fold selectivity for AT2Rs over AT1 receptors (AT1Rs). However, C21, particularly at high concentrations, has recently been shown to have AT2R-independent effects in the vasculature. We hypothesized that C21 induces natriuresis via AT2R translocation to the apical membrane and by activating a bradykinin (BK)-nitric oxide (NO)-cyclic GMP (cGMP) signaling pathway in the renal proximal tubule (RPT). In anesthetized, uninephrectomized, volume-expanded rats, intravenous (iv) C21 infusion (100, 200 and 300 ng/kg/min; each dose for 30 min) increased urinary Na+ excretion (UNaV) from 0.16±0.03 to 1.12±0.19, 1.51±0.08 and 2.03±0.21 μmol/min, respectively; all P <0.001). This natriuretic response was not altered by 24 h subcutaneous infusion of AT1R antagonist candesartan (CAND; 0.01 mg/kg/min), but was abolished by renal interstitial (RI) infusion of specific AT2R antagonist PD-123319 (PD). Blood pressure was unaltered by C21 or PD, but was lower in CAND-infused rats (P <0.0001). C21 infusion increased fractional excretion of Na+ (FENa) from 0.36±0.07 to 0.90±0.18% (P<0.05) and lithium (FELi) from 30.2±3.3 to 57.2±5.2% (P <0.01) without altering glomerular filtration rate or renal blood flow. Compared to control, C21 infusion increased RPT cell apical membrane AT2R protein (0.11±0.006 vs. 0.08±0.004 DU respectively) without changing total cell AT2R. C21-induced natriuresis was also accompanied by an increase in RI cGMP from 3.78±0.82 to 17.1±3.39 pmol/ml (P<0.01). The C21-induced increases in both UNaV and RI cGMP were abolished by RI infusion of NO synthase inhibitor L-NAME or BK B2 receptor antagonist icatibant. The results demonstrate that C21 initiates AT2R translocation to the RPT cell apical membrane inducing natriuresis in a BK-NO-cGMP-dependent manner in the absence of concurrent AT1R blockade. C21 holds promise as a natriuretic/diuretic agent for the treatment of fluid retaining states and hypertension.