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G protein-coupled receptors (GPCR) activate numerous intracellular signaling pathways. The oligomerization properties of GPCRs, and hence their cellular functions, may be modulated by various components within the cell membrane (such as the presence of cholesterol). Modulation may occur directly via specific interaction with the GPCR or indirectly by affecting the physical properties of the membrane. Here, we use pulsed Q-band double electron–electron resonance (DEER) spectroscopy to probe distances between R1 nitroxide spin labels attached to Cys163 and Cys344 of the β 1 -adrenergic receptor (β 1 AR) in n -dodecyl-β-D-maltoside micelles upon titration with two soluble cholesterol analogs, cholesteryl hemisuccinate (CHS) and sodium cholate. The former, like cholesterol, inserts itself into the lipid membrane, parallel to the phospholipid chains; the latter is aligned parallel to the surface of membranes. Global quantitative analysis of DEER echo curves upon titration of spin-labeled β 1 AR with CHS and sodium cholate reveal the following: CHS binds specifically to the β 1 AR monomer at a site close to the Cys163-R1 spin label with an equilibrium dissociation constant K D CHS ~1.4 ± 0.4 mM. While no direct binding of sodium cholate to the β 1 AR receptor was observed by DEER, sodium cholate induces specific β 1 AR dimerization ( K D cholate ~35 ± 6 mM and a Hill coefficient n ~ 2.5 ± 0.4) with intersubunit contacts between transmembrane helices 1 and 2 and helix 8. Analysis of the DEER data obtained upon the addition of CHS to the β 1 AR dimer in the presence of excess cholate results in dimer dissociation with species occupancies as predicted from the individual K D values.