Endothelin (ET) increases renal vascular resistance by constriction of post- and preglomerular vessels in the rat. However, ET receptor subtypes in renal microvessels have not been clearly defined. Radioligand binding experiments were performed in isolated arcuate and interlobular arteries as well as branching afferent arterioles to characterize 125I-ET-1 binding sites. Competitive inhibition assays were performed with ET-1, ET-2, ET-3, sarafotoxin 6b (S6b), BQ-123 (a preferential ETA receptor antagonist) and 4-Ala-ET-1 (a preferential ETB receptor agonist). Saturation data revealed a single class of high affinity binding sites with a kd of 0.31 +/- 0.03 nM and a Bmax of 1336 +/- 181 fmol/mg protein. Competitive inhibition of 125I-ET-1 binding showed that all assayed compounds displaced 125I-ET-1 in a dose-related manner. ET-1 displaced 100% of 125I-ET-1 binding, displaying monophasic curves with Bmax and kd values of 1369 +/- 170 fmol/mg protein and 0.35 +/- 0.04 nM, respectively. ET-2's displacement curves were similar to those of ET-1. ET-3 and S6b inhibited 100% of 125I-ET-1 binding in a biphasic manner, suggesting these peptides bind both high and low affinity sites. BQ-123 displaced about 50% of 125I-ET-1 in a monophasic manner, indicating a single high affinity binding site. 4-Ala-ET-1 displaced 125I-ET-1 in a clearly biphasic manner with an almost equal proportion of high and low affinity binding sites. Our results suggest that both ETA and ETB receptors are expressed in rat renal preglomerular vessels in almost equal proportions. However, the characteristics of competitive inhibition of 125I-ET-1 binding by several agents cannot be fully explained by a two-receptor model.