beta 1-adrenergic receptor (Adrb1) belongs to the superfamily of G-protein-coupled receptors (GPCRs) and plays a critical role in the regulation of heart rate and myocardial contraction force. GPCRs are phosphorylated at multiple sites to regulate distinct signal transduction pathways in different tissues. However, little is known about the location and function of distinct phosphorylation sites of Adrbl in vivo. To clarify the mechanisms underlying functional regulation associated with Adrbl phosphorylation in vivo, we aimed to identify Adrb1 phosphorylation sites in the mouse heart using phosphoproteomics techniques with nano-flow liquid chromatography/tandem mass spectrometry (LC-MS/MS). We revealed the phosphorylation residues of Adrb1 to be Ser274 and Ser280 in the third intracellular loop and Ser412, Ser417, Ser450, Ser451, and Ser462 at the C-terminus. We also found that phosphorylation at Ser274, Ser280, and Ser462 was enhanced in response to stimulation with an Adrbl agonist. This is the first study to identify Adrb1 phosphorylation sites in vivo. These findings will provide novel insights into the regulatory mechanisms mediated by Adrbl phosphorylation. (C) 2017 Elsevier Inc. All rights reserved.