Several different monomeric proteins with either one or two domains were used to study the effect of protein unfolding on effective hydrophobicity of proteins. Protein unfolding was inhibited by cross-linking with either toluene di-isocyanate or glutaraldehyde. The native enzyme was much more readily bound to the hydrophobic resin than the cross-linked species. The ligand, 3-phosphoglycerate, significantly decreased the amount of phosphoglycerate kinase able to bind to octyl-Sepharose. Sucrose also caused a statistically significant decrease in protein binding to the hydrophobic resin. These studies support the concept that the degree of protein unfolding influences effective hydrophobicity as measured by retention on octyl-Sepharose.