Chromatographic properties of four cholesterol bonded phases with different structures were studied. The columns used were packed with a stationary phase containing a cholesterol molecule attached to the silica surface using different types of linkage molecules. Columns were compared according to the retention and separation selectivity of steroid hormones. The measurements were done using binary hydro-organic mobile phases with methanol, ethanol and acetonitrile as an organic modifier. The presented results show that the coverage density of the bonded ligands and the type of organic modifier strongly influence the retention mechanism and separation selectivity of steroid hormones on cholesterol-containing adsorbents.