The competitive binding activities of a number of angiotensin analogues were determined in a radioligand-receptor assay employing bovine adrenal crotex homogenate and [125I] iodoangiotensin II. This assay system has been shown to provide a precise and convenient method for evaluation and comparison of the binding-inhibition potencies of angiotensin II derivatives with agonist and antagonist activities. Agonist analogues of angiotensin II showed competitive binding activities in proportion to their known biological activities upon aldosterone production by the adrenal zona glomerulosa. Thus, the [Des-Asp1] heptapeptide of angiotensin II was equipotent with the native octapeptide in terms of binding-inhibition activity, and the [Sar1] derivative of angiotensin II was more potent than the native peptide. By contrast, the [Des-Asp1, Des-Arg2] hexapeptide and the [Des-Phe8] heptapeptide showed less than 1% of the activity of angiotensin II. Angiotensin II antagonists formed by C-terminal substitution of the octapeptide with isoleucine or alanine were also found to exhibit binding-inhibition activities in proportion to their known potencies as antagonists of the action of angiotensin II upon smooth muscle. Certain antagonists, such as [1-guanidoacetic, 8-isoleucine]-angiotensin II and [1-sarcosine, 8-isoleucine]-angiotensin II displayed significantly greater binding-inhibition potency than E1Asp1, Ileu5,-angiotensin II in the adrenal receptor system. Several, such as [Sar1, Ala8]-angiotensin II and [MeAla1, Ileu8]-antiotensin II were approximately equipotent with angiotensin II, while others such as [1-succinic acid, 8-alanine]-angiotensin II and [D-Ileu8]-angiotensin II showed no significant binding-inhibition activity. The relative binding-inhibition potencies of the antagonist peptides showed a general correlation with the pA2 values of the same components determined upon the smooth muscle response of the isolated aortic strip. The binding-inhibition potency of angiotensin II antagonists was also strongly influenced by the charge of the N-terminal residue, with enhancement of activity by more basic substituents and abolition of activity by highly acidic residues. The influence of the basicity of the N-terminus upon receptor binding was also observed with the agonist analogue [Sar1]-angiotensin II, which showed a 2- to 3-fold increase in binding-inhibition potency in comparison to native angiotensin II. The significant enhancement of binding-inhibition potency by N-terminal sarcosine substitution is attributable to higher affinity of the modified peptide for the angiotensin II receptor site, and is consistent with the increased activity of [Sar1]-angiotensin II upon smooth muscle and aldosterone production in vitro. The determination of binding-inhibition activity in the adrenal radioligand receptor assay provides a valid and convenient method for analysis of the role of binding affinity in the actions of competitive antagonists upon the responses of target cells to angiotensin II.