A new 1,3-alternate 25,27-dipropoxy-26,28-bis-[3-propyloxy]-calix[4]arene-bonded silica gel stationary phase (1,3-Alt CalixPr) has been prepared and used for the separation of di- and trisubstituted aromatic positional isomers by HPLC. The effect of organic modifier content, pH and column temperature on retention and selectivity of the benzene derivatives were studied. The retention mechanism was also discussed. The results indicated that the stationary phase behaves like a reversed-phase packing. However inclusion, hydrophobic, hydrogen bonding and pi-pi interactions seem to be involved in separation process.