Abstract Hydrophobic interaction chromatography of proteins and peptides on Spheron P-300 has been studied. Proteins such as human serum albumin, chymotrypsinogen and lysozyme are retained by this support; the higher the concentration of the salt added, the more protein is retained. The sorption and desorption were examined quantitatively with lysozyme. The theoretical plate height and its dependence on temperature during hydrophobic interaction chromatography of the lysozyme were determined. The hydrophobic interaction of the support was demonstrated by hydrophobic interaction chromatography of the protein components of human serum proteins, hog pancreatic amylase extract, and a peptide mixture obtained from tryptic digests of lysozyme. The effect of pH on the elution of human serum albumin, chymotrypsinogen and lysozyme was observed. The fractionation of crude hog pancreatic amylase by gradient elution was used to demonstrate the effect of alcohols as polarity-reducing agents. The properties required of materials used for hydrophobic interaction chromatography of biopolymers are discussed.