The effect of cholesterol on the properties of lipid membranes has been investigated by computer simulations. For this purpose, the crossmembrane free energy profiles of eight penetrants, i.e., H2O, O-2, CO, CO2, NO, NH3, CHCl3, and formamide, have been calculated by the cavity insertion Widom (CIW) method in four simulated dimyristoylphosphatidylcholine (DMPC)/cholesterol mixed membranes of different compositions, i.e., containing 0%, 4%, 8%, and 40% cholesterol. The compositions of the simulated two component membranes have been selected from both sides of the DMPC/cholesterol miscibility gap, and the pure DMPC membrane has been regarded as a reference system. It is found that cholesterol increases the amount of spherical cavities in the membrane region in which their OH group is located and, hence, lowers the solvation free energy of the penetrants in this region. For strongly polar solutes, this is the region of the minimum of the free energy profiles, and hence, by lowering this minimum, cholesterol increases the free energy barrier of the crossmembrane transport of such penetrants. On the other hand, for larger and apolar or moderately polar solutes, such as CO2 and CHCl3, the free energy profiles exhibit a peak in this region. In the case of CHCl3, cholesterol is found to lower and, above a certain concentration, eliminate this peak and thus considerably decrease the free energy barrier of the crossmembrane transport of this molecule. On the other hand, in the case of CO2, this peak is transformed to a dip by cholesterol, and hence, the free energy barrier of the crossmembrane penetration of CO2 is first lowered by increasing the fraction of cholesterol in the membrane up to a certain concentration, above which a further increase of the amount of cholesterol results in an increasing free energy barrier. Finally, in the case of the diatomic penetrants, neither the maximum nor the minimum of the free energy profiles is located in the region where cholesterol lowers the solvation free energy, and thus, the free energy barrier of the crossmembrane transport of these molecules is not affected by cholesterol.