We have previously shown that individual β-amino acid substitution in angiotensin (Ang) II reduced Ang II type 1 receptor (AT1R) but not Ang II type 2 receptor (AT2R)-binding and that the heptapeptide Ang III exhibited greater AT2R:AT1R selectivity than Ang II. Therefore, we hypothesized that β-amino-acid-substituted Ang III peptide analogues would yield highly selective AT2R ligands, which we have tested in binding and functional vascular assays. In competition binding experiments using either AT1R- or AT2R-transfected human embryonic kidney (HEK)-293 cells, novel β-substituted Ang III analogues lacked appreciable AT1R affinity, whereas most compounds could fully displace (125)I-Sar(1)Ile(8) Ang II from AT2R. The rank order of affinity at AT2R was CGP42112 > Ang III > β-Pro(7) Ang III=Ang II > β-Tyr(4) Ang III ≥ PD123319 >> β-Phe(8) Ang III >> β Arg(2) Ang III=β-Val(3) Ang III >> β-Ile(5) Ang III. The novel analogue β-Pro(7) Ang III was the most selective AT2R ligand tested, which was >20,000-fold more selective for AT2R than AT1R. IC50 values at AT2R from binding studies correlated with maximum vasorelaxation in mouse aortic rings. Given that β-Pro(7) Ang III was an AT2R agonist, we compared β-Pro(7) Ang III and native Ang III for their ability to reduce blood pressure in separate groups of conscious spontaneously hypertensive rats. Whereas Ang III alone increased mean arterial pressure (MAP), β-Pro(7) Ang III had no effect. During low-level AT1R blockade, both Ang III and β-Pro(7) Ang III, but not Ang II, lowered MAP (by ∼30 mmHg) at equimolar infusions (150 pmol/kg/min for 4 h) and these depressor effects were abolished by the co-administration of the AT2R antagonist PD123319. Thus, β-Pro(7) Ang III has remarkable AT2R selectivity determined in binding and functional studies and will be a valuable research tool for insight into AT2R function and for future drug development.