A hallmark of G-protein-coupled receptors (GPCRs) is the conversion of external stimuli into specific cellular responses. In this tightly-regulated process, extracellular ligand binding by GPCRs promotes specific conformational changes within the seven transmembrane helices, leading to the coupling and activation of intracellular "transducer" proteins, such as heterotrimeric G proteins. Much of our understanding of the molecular mechanisms that govern GPCR activation is derived from experiments with purified receptors reconstituted in detergent micelles. To elucidate the influence of the phospholipid bilayer on GPCR activation, here we interrogated the functional, pharmacological, and biophysical properties of a GPCR, the β-adrenergic receptor (βAR), in high-density lipoprotein (HDL) particles. Compared with detergent-reconstituted βAR, the βAR in HDL particles had greatly enhanced levels of basal (constitutive) activity and displayed increased sensitivity to agonist activation, as assessed by activation of heterotrimeric G protein and allosteric coupling between the ligand-binding and transducer-binding pockets. Using F NMR spectroscopy, we directly linked these functional differences in detergent- and HDL-reconstituted βAR to a change in the equilibrium between inactive and active receptor states. The contrast between the low levels of βAR constitutive activity in cells and the high constitutive activity observed in an isolated phospholipid bilayer indicates that βAR basal activity depends on the reconstitution system and further suggests that various cellular mechanisms suppress βAR basal activity physiologically. Our findings provide critical additional insights into GPCR activation and reveal how dramatically reconstitution systems can impact membrane protein function.