While the new Martini 3 cholesterol model was initially developed and implemented in GROMACS, it has also been successfully ported to OpenMM. During the implementation of the Martini 2 cholesterol model in OpenMM , difficulties were encountered with the software's algorithm for handling topology constraints . Unlike GROMACS, which utilizes the LINCS algorithm , OpenMM adopts the Constant Constraint Matrix Approximation (CCMA) for constraint treatment . This approach involves the construction of a matrix that characterizes the coupling between constraints based on bonds and angles in the topology. For any pair of constraints that share an atom, a triangle defining an angle, bond, or third constraint is essential in determining the coupling between them according to the CCMA algorithm. Consequently, the topology of the Martini 2 cholesterol model, which features two triangular frames of constraints sharing an edge, causes instability with the CCMA algorithm. Along with the improvements already described, the bonded topology used in the new Martini 3 cholesterol model also ensures compatibility with the CCMA constraint algorithm within the OpenMM platform. By using a single-frame virtual site approach for the topology, which relies on a single triangle of constraints, the coupling between elements in the constraint matrix in the CCMA algorithm can be properly defined, thereby ensuring its stability. The accuracy of the implementation of the new cholesterol model in OpenMM was evaluated using two approaches: i-single-point calculations; and ii-determination of membrane biophysical properties in equilibrium in lipid mixtures with increasing cholesterol concentrations.