Abstract A column-switching high-performance liquid chromatographic (CS-HPLC) system which consisted of an anhydrotrypsin (AHT)-immobilized diol—silica precolumn and a reversed-phase analytical column was developed for the selective separation of peptides having Arg or Lys at their C-termini. Tuftsin (Thr-Lys-Pro-Arg) could be enriched almost quantitatively on the precolumn when loadd with water as a carrier solvent and the precolum was washed with 10–30 m M acetate buffer (pH 5.0). An investigation of the affinity characteristics of 55 peptides to the AHT precolumn showed that among twelve peptides having Arg or ArgNH 2 at their C-termini and more than four amino acid residues, ten were retained almost quantitatively on the precolumn, and eight out of nine peptides having Lys at their C-termini were less retained. The peptide having d-Arg at its C-termini was not retained. However, twelve out of thirty peptides having no Arg or Lys at their C-termini were also retained, but the retention was greatly decreased, in contrast to the Arg peptides, when the precolumn was washed with 20m M calcium chloride solution. The results indicate that the CS-HPLC system equipped with an AHT precolumn offers new selectivity in the HPLC separation of peptides.