Estradiol (E-2) and E-2 oleate associate with high-density lipoproteins (HDLs). Their orientation in HDLs is unknown. We studied the orientation of E-2 and E-2 oleate in membranes and reconstituted HDLs, finding that E-2 and E-2 oleate are membrane-associated and highly mobile. Our combination of NMR measurements, molecular dynamics simulation, and analytic theory identifies three major conformations where the long axis of E-2 assumes a parallel, perpendicular, or antiparallel orientation relative to the membrane's z-direction. The perpendicular orientation is preferred, and furthermore, in this orientation, E-2 strongly favors a particular roll angle, facing the membrane with carbons 6, 7, 15, and 16, whereas carbons 1, 2, 11, and 12 point toward the aqueous phase. In contrast, the long axis of E-2 oleate is almost exclusively oriented at an angle of similar to 60 degrees to the z-direction. In such an orientation, the oleoyl chain is firmly inserted into the membrane. Thus, both E-2 and E-2 oleate have a preference for interface localization in the membrane. These orientations were also found in HDL discs, suggesting that only lipid-E-2 interactions determine the localization of the molecule. The structural mapping of E-2 and E-2 oleate may provide a design platform for specific E-2-HDL-targeted pharmacological therapies.