Our previous studies have demonstrated that angiotensin (Ang) type-2 receptors (AT2R) mediate natriuresis by inhibiting Na+ reabsorption in the renal proximal tubule and that des1-aspartyl-Ang II (Ang III) is the preferred endogenous agonist for this response. Natriuretic responses to intrarenal Ang III infusion do not occur in the absence of concurrent systemic Ang type-1 receptor (AT1R) blockade or aminopeptidase N inhibition to prevent Ang III degradation. In the present study, we hypothesize that Compound 21 (C21), a highly selective non-peptide AT2R agonist, administered chronically in the absence of concurrent AT1R blockade can induce natriuresis in wild-type mice (WT; C57B6) and that this response does not occur in AT2R-null mice (AT2-null). WT and AT2-null (N=8/group) on normal diet (0.3% NaCl) were infused subcutaneously with C21 (300 ng/kg/min) or vehicle (V) 5% dextrose in water via osmotic mini-pump continuously for 7 days. Food/fluid intake and systolic blood pressure (SBP; via telemetry for 30 min at 8 am) were monitored daily and 24 h urine Na+ excretion (UNaV) was measured at baseline and infusion days 1, 4, and 7. Baseline UNaV in V-infused WT was 0.09 ± 0.01 μmol/min and did not change with V infusion. In C21-infused WT, UNaV increased from a baseline of 0.11 ± 0.01 to 0.13 ± 0.02 μmol/min on day 4 and to 0.17 ± 0.02 μmol/min on day 7 (F=15.0, P<0.0001 from V-infused WT and AT2-null). UNaV in both V- and C21-infused AT2-null decreased in parallel from a baseline of 0.11 ± 0.01 to 0.07 ± 0.01 μmol/min on day 4 and to 0.06 ± 0.01 μmol/min on day 7 (P<0.0001 from C21-infused WT). Dietary consumption was identical among the 4 groups of mice over the 7 day study. SBP was similar in the 4 groups during the control period. The results demonstrate that C21 induces natriuresis chronically in an AT2R-dependent manner in the absence of AT1R blockade and indicates that C21 may be a useful natriuretic/diuretic agent in disorders of fluid retention.