The effects of the pH, organic modifiers and buffer salt of the mobile phase upon the retention and separation characteristics of avidin- and ovomucoid-bonded silica columns as proteinous chiral stationary phases for HPLC were investigated. The enantioselectivity was greatly affected by changing the mobile-phase composition, possibly because of induced alterations in the protein conformation. Hydrophobic and Coulombic interactions play major roles in the retention, and there are some similarities between the avidin column and the ovomucoid column. However, the stereoselectivity does not necessarily reflect the retention processes on these columns. No significant decrease in the retention or enantioselectivity was observed, even after the injection of 200 samples.