In order to generate antibodies suitable for immunological studies on β-adrenoceptors constitutively expressed at low levels in cells or tissues we have produced fusion proteins of the amino- and carboxy-terminus, and the second extracellular loop of the human β₁- or β₂-adrenoceptors with bacterial glutathione-S-transferase in E. coli. Rabbit antibodies raised against these fusion proteins strongly reacted with intact human β₁- or β₂-adrenoceptors in a subtype- and domain-specific manner. Antibodies directed against the second extracellular loop of the β₁-adrenoceptor reacted stronger with non-denatured receptors and decreased the affinity of the ³H-labelled antagonist (−)-4-(3-t-butylamino-2-hydroxypropoxy)-[5,7-³H]benzimidazol-2-one ([³H]CGP 12 177), indicating a specific interaction with the native receptor. In contrast, antibodies directed against carboxy- and amino-terminal receptor domains reacted strongly both with denatured and non-denatured receptors but did not interfere with binding of [³H]CGP 12 177. Affinity purified antibodies were used for detecting the β₁- or the β₂-adrenoceptor subtype heterologously produced in Sf9 cells by enzyme-linked immunosorbent assay, Western blotting, immunoprecipitation, and indirect immunofluorescence microscopy. Moreover, we could demonstrate that avidity, titers, and specificity of these antibodies were high enough for studying β-adrenoceptors constitutively expressed in human A431 cells, where we observed a patched membrane distribution of the receptors.