The study reports the 2.7 Å resolution crystal structure of a β1-adrenergic receptor (β1AR) complexed with the high-affinity antagonist cyanopindolol. The modified turkey β1AR, selected for its antagonist conformation and improved thermostability, was used. The ligand-binding pocket is defined by 15 amino acid residues from four transmembrane α-helices and extracellular loop 2 (EL2). This loop, stabilized by disulfide bonds and a sodium ion, forms the entrance to the pocket. Cyanopindolol binding to β1AR involves similar interactions to carazolol binding to β2AR. A unique short α-helix in EL2 interacts with the conserved DRY motif in helix 3, which is essential for receptor activation. The structure provides insights into subtype specificity and ligand selectivity, highlighting differences in residues near the binding pocket that affect ligand binding. The β1AR structure also reveals a 2-3 Å tightening of the pocket upon agonist binding, consistent with the activation mechanism.The study reports the 2.7 Å resolution crystal structure of a β1-adrenergic receptor (β1AR) complexed with the high-affinity antagonist cyanopindolol. The modified turkey β1AR, selected for its antagonist conformation and improved thermostability, was used. The ligand-binding pocket is defined by 15 amino acid residues from four transmembrane α-helices and extracellular loop 2 (EL2). This loop, stabilized by disulfide bonds and a sodium ion, forms the entrance to the pocket. Cyanopindolol binding to β1AR involves similar interactions to carazolol binding to β2AR. A unique short α-helix in EL2 interacts with the conserved DRY motif in helix 3, which is essential for receptor activation. The structure provides insights into subtype specificity and ligand selectivity, highlighting differences in residues near the binding pocket that affect ligand binding. The β1AR structure also reveals a 2-3 Å tightening of the pocket upon agonist binding, consistent with the activation mechanism.