Endothelin B receptor (ETBR) is a G protein-coupled receptor that mediates a variety of signals by binding to vasoconstrictive peptides, endothelins. Monoclonal antibodies were prepared against human ETBR using the full-length protein expressed in Sf9 cells. Five typical monoclonal antibodies were characterized further for their recognition. The epitopes for the 2A5, 9A3 and 21A1 antibodies were mapped within the N-terminal extracellular sequences, V71-I85 and E27-Q41, respectively, which differ between the human and mouse ET(B)Rs. All of these antibodies labeled cell surface ETBR expressed in COS cells, suggesting that their recognition sites exist in the extracellular domain. In addition, the immobilized antibodies could purify ETBR expressed in Sf9 cells to the majority under mild conditions. Thus, immunization with the recombinant full-length membrane protein provides a strategy to produce monoclonal antibodies recognizing the native protein.