The analysis of membrane receptors for hormones and neurotransmitters has progressed considerably by pharmacological and biochemical means and more recently through the use of specific antibodies. To generate and characterize a moloclonal antibody against β-adrenergic receptor, a synthetic β2-adrenergic receptor peptide (Phe-Gly-Asn-Phe-Trp-Cys-Phe-Trp-Thr-Ser-Ile-Asp-Val-Leu) which may comprise part of β-adrenergic receptor ligand binding pocket was coupled to Keyhole Limpet Hemocyanin (KLH) and used as an immunogen. Male BALB/C mice were immunized with this antigen and the immunized spleen was fused with myeloma SP2/0-Ag14 cells to produce monoclonal antibodies. Two clones were obtained but one of monoclonal antibodies, mAb5G09, was used throughout in this study because the other clone, mAb5A11 showed weak immunoreactivity against KLH as well. The mouse monoclonal antibody mAb5G09 produced in this study showed immunoreactivity to peptide-KLH conjugates and also to human A431 cells and guinea pig lung β2-adrenergic receptor as revealed by ELISA and western blot. In the course of determination of the effects of mAb5G09 on β-receptor ligand binding, it was observed that mAb5G09 specifically bound β-adrenergic radioligand [³H]dihydroalprenolol (DHA) with a dissociation constant (Kd) of 60 nM. The [³H]DHA binding activity of mAb5G09 had characteristics of immunoglobulins and the binding activity was not observed in the control anti-KLH monoclonal antibody. The monoclonal antibody, mAb5G09 produced in this study may provide useful models for the study of the structure of receptor binding sites.