We investigated the characteristics of 125I-AII binding to rat adrenal and bovine cerebellar membranes in the presence and absence of new nonpeptide angiotensin II (AII) receptor ligands. The imidazole AII ligands, DUP753 and WL19, both produced biphasic competition curves to 125I-AII binding in rat adrenal glomerulosa and adrenal medulla particles, suggesting the existence of two distinct AII binding sites. Antagonist affinity (Ki) and binding capacity (Bmax) for each binding site was determined using nonlinear analysis of competition data fit to a two-site model. The high capacity site (68% of total specific 125I-AII bound) in glomerulosa had high affinity for DUP753 (4.6 +/- 0.8 nM) and low affinity for WL19 (29 +/- 3 microM), and the low capacity site had high affinity for WL19 (3.3 +/- 1.4 nM) and low affinity for DUP753 (51 +/- 9 microM). Conversely, in medulla, the high capacity site (77% total binding) had high affinity for WL19 (19 +/- 6 nM) and low affinity for DUP753 (29 +/- 8 microM), and the low capacity site had low affinity for WL19 (25 +/- 7 microM) but a high affinity for DUP753 (2.8 +/- 2.0 nM). In glomerulosa, binding parameters for the nonpeptide ligands at each site derived from monophasic competition curves obtained in the presence of either 0.3 microM DUP753 or WL19 to selectively block the high or low capacity binding site, respectively, were similar to values determined from the biphasic competition curves. Unlike the nonpeptide inhibitors, unlabeled AII yielded monophasic inhibition curves.(ABSTRACT TRUNCATED AT 250 WORDS)