The second factor impacting this population was the miscibility of the cholesterol hydroxyl beads within the phosphate/glycerol (PO4/GL) layer. Using a P1 bead for the hydroxyl moiety, as recommended per Martini 3 guidelines, led to slightly decreased miscibility within the PO4/GL layer compared to Martini 2. If we compare free energies of mixing between the two force field iterations, this is mostly due to decreased miscibility of Martini 3 P1 beads with SN4a and N4a glycerol beads, which seems in line with small molecule miscibility of alcohols and esters . Increasing (P1 to P2) or decreasing (P1 to N6) the polarity of the ROH bead had no beneficial effect. While P2 has stronger interaction towards the phosphodiester Q5, it is even less interacting with the glycerol N4a beads. Similarly, while N6 maintains the interaction with Q5 and increases it slightly with N4a, it is also less repulsive towards the acyl chains composed of C1 beads, facilitating the ingress of cholesterol. While the overall interaction balance in Martini 3 is now better than in Martini 2, looking at the free energy of insertion of cholesterol in a POPC bilayer (Supplementary Figure ) we do see an underestimated barrier between cholesterol ROH inserted at the PO4/GL depth and in the center of the membrane, when compared to both Martini 2 (~ 2 kJ/mol difference) and atomistic references (~ 15 kJ/mol difference) . This explains the increase in flip flop rates in Martini 3, compared to its previous iteration. However, the ~ 30 kJ/mol discrepancy between Martini 2 and atomistic references when cholesterol is fully inserted in the center plane has now been improved in Martini 3 (~ 5 kJ/mol). Additionally, the free energy of cholesterol insertion at its equilibrium position has also been slightly improved, with a decrease in the discrepancy from ~ 20 kJ/mol in Martini 2 to ~ 10 kJ/mol in Martini 3. These improvements, while still not perfect, showcase the new model's ameliorated hydrophobicity profile, which will allow users to more easily explore cholesterol-associated features that were otherwise compromised by its overestimated hydrophobicity.