Phase diagrams were determined for aqueous two-phase systems (ATPSs) formed by different paired combinations of Dextran (Dex-75), Ficoll-70, polyethylene glycol (PEG-8000), hydroxypropyl starch (PES-100), and Ucon50HB5100 (a random copolymer of ethylene glycol and propylene glycol) all containing 0.15M NaCl in 0.01M phosphate buffer, pH 7.4, at 23 degrees C. Partition coefficients of a series of dinitrophenylated (DNP) amino acids with aliphatic side-chains were studied in all the ATPSs at particular polymer concentrations. Free energies of transfer of a methylene group between the coexisting phases, DeltaG(CH(2)), were determined as measures of the difference between the hydrophobic character of the phases. Furthermore, partition coefficients of tryptophan (Trp) and its di- and tri-peptides and a set of p-nitrophenyl (NP)-monosaccharides were measured in all the two-phase systems, and the data obtained compared with the DeltaG(CH(2)) values obtained in the systems. It was established that for eight out of 10 of two-phase systems of different polymer compositions the partition coefficients for Trp peptides correlate well with the DeltaG(CH(2)) values. Similar correlations for NP-monosaccharides were valid for seven out of 10 two-phase systems. These observations indicate that the difference between the hydrophobic characters of the coexisting phases represented by the DeltaG(CH(2)) value cannot be used as a single universal measure for comparison of the ATPSs of different polymer compositions.