Partition of the natural dye carmine has been studied in aqueous two-phase systems prepared by mixing aqueous solutions of polymer or copolymer with aqueous salt solutions (Na(2)SO(4) and Li(2)SO(4)). The carmine dye partition coefficient was investigated as a function of system pH, polymer molar mass, hydrophobicity, system tie-line length and nature of the electrolyte. It has been observed that the carmine partition coefficient is highly dependent on the electrolyte nature and pH of the system, reaching values as high as 300, indicating the high potential of the two-phase extraction with ATPS in the purification of carmine dye. The partition relative order was Li(2)SO(4)"Na(2)SO(4). Carmine molecules were concentrated in the polymer-rich phase, indicating an enthalpic specific interaction between carmine and the pseudopolycation, which is formed by cation adsorption along the macromolecule chain. When the enthalpic carmine-pseudopolycation interaction decreases, entropic forces dominate the natural dye-transfer process, and the carmine partitioning coefficient decreases. The optimization of the extraction process was obtained by a central composite face-centered (CCF) design. The CCF design was used to evaluate the influence of Li(2)SO(4) and PEO 1500 concentration and of the pH on the partition coefficient of carmine. The conditions that maximize the partition of carmine into the top phase were determined to be high concentrations of PEO and Li(2)SO(4) and low pH values within the ranges studied.