Abstract The chromatographic behavior of some nucleosides, pyrimidines and purines on a new p-tert-butyl-calixarene-bonded silica gel stationary phase (CABS) were studied by high performance liquid chromatography. Their retention behavior on CABS were compared with those on ODS. The influence of mobile phase variables, such as methanol content, pH and ionic strength on the retention behavior were studied. Some nucleosides, pyrimidines and purines on CABS were successfully separated. The results show that the calixarene-bonded phase exhibits high selectivities for the above analytes in high aqueous mobile phases. According to the chromatographic data, it is indicated that hydrophobic interaction, hydrogen-bonding interaction, and dipole–dipole interaction are mainly responsible for the retention behavior. In addition, in some extent, the vertical stacking action of the analytes on CABS can also change the retention behavior. CABS was superior to ODS in the routine fast separation of nucleosides and bases.