In fully saturated lipid bilayers, the Martini 3 cholesterol is inserted in the membrane in its canonical upright orientation, with the hydroxyl group just protruding into the phosphodiester region of the bilayer (Figure ), and oriented perpendicular to the bilayer plane (Supplementary Figure ). This canonical insertion pose matches both atomistic and Martini 2 behavior, as well as experimental data on cholesterol membrane orientation 102 . By decreasing the lipid bilayer saturation, a minor population of cholesterol molecules, which transiently reside in the center of the membrane and are oriented parallel to the membrane plane, begin appearing in our Martini 3 simulations (Figure a; Supplementary Figure ). This population accounts for a very minor part of the total cholesterol content in mono-unsaturated lipid bilayers, and gradually increases with decreasing saturation, becoming the prevalent insertion pose in poly-unsaturated DAPC lipid bilayers.