The structure of the β1-adrenergic G protein-coupled receptor (GPCR) in complex with the high-affinity antagonist cyanopindolol has been determined at 2.7 Å resolution. This study provides insights into the structural basis of GPCR function and drug binding. The receptor, derived from turkey, was engineered for enhanced thermostability and antagonist conformation. The ligand-binding pocket is formed by amino acid residues in four transmembrane α-helices and extracellular loop 2, which is stabilized by two disulphide bonds and a sodium ion. Cyanopindolol and carazolol bind to β1 and β2 receptors, respectively, through similar interactions. A short helix in cytoplasmic loop 2 interacts with the DRY motif in helix 3, essential for receptor activation. GPCRs are integral membrane proteins involved in signal transduction. The β1 and β2 adrenergic receptors are key GPCRs in the β family, with significant sequence identity. The structure of β1-AR reveals similarities to β2-AR, with minor differences in conformation. The β1-AR structure shows the receptor in an inactive state, complexed with an antagonist, and provides a framework for understanding subtype-specific drug binding. The extracellular loops are highly conserved, with the sodium ion playing a stabilizing role. The cytoplasmic loop 2 forms a short α-helix, which is not present in β2-AR or rhodopsin, and interacts with the DRY motif. The structure highlights the role of the second intracellular loop in G protein coupling, with the DRY motif being crucial for activation. The β1-AR structure also reveals the presence of a salt bridge, termed the "ionic lock," which may be involved in maintaining the inactive state of GPCRs. However, this salt bridge is not present in the β1-AR or β2-AR structures, suggesting it may not be essential for the inactive state. The ligand-binding pocket of β1-AR contains 15 amino acid residues that interact with cyanopindolol. These residues are identical to those in human β2-AR, indicating similar binding mechanisms. Differences in amino acid residues near the ligand-binding pocket between β1-AR and β2-AR may explain differences in ligand selectivity. The structure provides a basis for understanding the selectivity of β-agonists and antagonists, and highlights the importance of the second extracellular loop in ligand binding and G protein coupling. The study also demonstrates the challenges in crystallizing GPCRs, which are often unstable and prone to conformational changes. The β1-AR structure, determined using molecular replacement, provides a detailed view of the receptor's structure and function, contributing to the understanding of GPCR mechanisms and drug development. The findings have implications for the designThe structure of the β1-adrenergic G protein-coupled receptor (GPCR) in complex with the high-affinity antagonist cyanopindolol has been determined at 2.7 Å resolution. This study provides insights into the structural basis of GPCR function and drug binding. The receptor, derived from turkey, was engineered for enhanced thermostability and antagonist conformation. The ligand-binding pocket is formed by amino acid residues in four transmembrane α-helices and extracellular loop 2, which is stabilized by two disulphide bonds and a sodium ion. Cyanopindolol and carazolol bind to β1 and β2 receptors, respectively, through similar interactions. A short helix in cytoplasmic loop 2 interacts with the DRY motif in helix 3, essential for receptor activation. GPCRs are integral membrane proteins involved in signal transduction. The β1 and β2 adrenergic receptors are key GPCRs in the β family, with significant sequence identity. The structure of β1-AR reveals similarities to β2-AR, with minor differences in conformation. The β1-AR structure shows the receptor in an inactive state, complexed with an antagonist, and provides a framework for understanding subtype-specific drug binding. The extracellular loops are highly conserved, with the sodium ion playing a stabilizing role. The cytoplasmic loop 2 forms a short α-helix, which is not present in β2-AR or rhodopsin, and interacts with the DRY motif. The structure highlights the role of the second intracellular loop in G protein coupling, with the DRY motif being crucial for activation. The β1-AR structure also reveals the presence of a salt bridge, termed the "ionic lock," which may be involved in maintaining the inactive state of GPCRs. However, this salt bridge is not present in the β1-AR or β2-AR structures, suggesting it may not be essential for the inactive state. The ligand-binding pocket of β1-AR contains 15 amino acid residues that interact with cyanopindolol. These residues are identical to those in human β2-AR, indicating similar binding mechanisms. Differences in amino acid residues near the ligand-binding pocket between β1-AR and β2-AR may explain differences in ligand selectivity. The structure provides a basis for understanding the selectivity of β-agonists and antagonists, and highlights the importance of the second extracellular loop in ligand binding and G protein coupling. The study also demonstrates the challenges in crystallizing GPCRs, which are often unstable and prone to conformational changes. The β1-AR structure, determined using molecular replacement, provides a detailed view of the receptor's structure and function, contributing to the understanding of GPCR mechanisms and drug development. The findings have implications for the design