One of many important roles of the naturally occurring nonapeptide bradykinin (BK) is the modulation of vascular tone. As endothelial cells may play vital roles in the physiology of vascular tissues, it is important to understand the interaction of this peptide with endothelial cells. By using radiolabeled BK we have demonstrated for the first time BK specific binding sites in membranes of bovine pulmonary artery endothelial cells. There are two types of receptors shown in binding assay: one with a saturable, high-affinity binding (Kd = 1.28 +/- 0.21 nM, maximum binding = 111.4 +/- 12.0 fmol/mg) and the other with not readily saturable, low-affinity binding. The activation of BK receptors on bovine pulmonary artery endothelial cells is assessed by two functional assays, namely, the release of endothelium-derived relaxing factor and the elevation of cytosolic calcium. The characteristics of the BK receptors in both binding and functional assays indicate that the high-affinity binding is to B2 receptors and the low-affinity binding is to B1 receptors on the cells. Thus, high-affinity binding is blocked by B2 antagonists, D-Arg[Hyp3, thienylalanine5,8,D-Phe7]-BK and [thienylalanine5,8, D-Phe7]-BK and low-affinity binding is blocked by B1 antagonist des-Arg9[Leu8]-BK. The elevation of intracellular calcium and the release of endothelium-derived relaxing factor in response to BK in endothelial cells is predominately through B2 receptor activation. The ability to subtype BK receptors in endothelial cells may facilitate the understanding of the vascular functions of BK and the potential design of drugs to regulate these functions.