The human β₁-adrenoceptor is an immune target for autoantibodies with functional activity in cardiovascular diseases. Different epitopes on the extracellular domains of the receptor are involved. To study the immunological and pharmacological properties of these epitopes, rabbits were immunized with peptides corresponding to a large domain in the N-terminal part of the receptor and to its first and second extracellular loops. In contrast to the two other peptides, the first extracellular loop did not have immunogenic properties but acted as a hapten. Antibodies affinity-purified with the three synthetic peptides were able to significantly immunoprecipitate the solubilized receptor, confirming that they recognized the target receptor. While antibodies against the N-terminal domain did not inhibit the binding of a radiolabelled antagonist to the receptor, those against the first and second extracellular loop showed non-competitive inhibition. Similarly, only the two latter antibodies exerted a specific agonist-like effect on the receptor, as assessed on neonatal rat cardiomyocytes in culture. Our results are in accordance with those found for human anti-receptor autoantibodies with functional effects. We conclude that not all extracellular epitopes give rise to functional autoantibodies with potential physiopathological relevance in cardiac diseases with an autoimmune component.