Affinity partitioning of enzymes and plasma proteins in aqueous two-phase systems has been reviewed. Besides basic theoretical considerations of the principle of affinity partitioning the chemistry of coupling ligands to the polymers, the nature and properties of selected biomimetic ligands like dye-ligands, immunoligands, metal chelate ligands and hydrophobic ligands are reported. The usefulness of affinity partitioning for studying the affinity of ligands and their specificity to proteins has been demonstrated by selected examples. The method proved also applicable to study the structural dynamics of proteins as exemplified with phosphofructokinase from baker's yeast and human alpha-2-macroglobulin. The current knowledge of metal chelate affinity partitioning is presented as well as the applicability of affinity partitioning for the purification of enzymes.