We have previously shown that individual beta-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 beta-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 beta-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 > beta-Pro(7) Ang III=Ang II > beta-Tyr(4) Ang III >/= PD123319 >> beta-Phe(8) Ang III >> beta Arg(2) Ang III=beta-Val(3) Ang III >> beta-Ile(5) Ang III. The novel analogue beta-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 beta-Pro(7) Ang III was an AT2R agonist, we compared beta-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), beta-Pro(7) Ang III had no effect. During low-level AT1R blockade, both Ang III and beta-Pro(7) Ang III, but not Ang II, lowered MAP (by approximately 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, beta-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.