The hydrophobicity of five proteins was estimated by reversed-phase chromatography (RPC), hydrophobic-interaction chromatography (HIC), and precipitation with ammonium sulfate. These data were correlated to partition behavior in aqueous two-phase systems. A parameter (1/m*) that was derived from the precipitation curves is based on the solubility of proteins in an electrolyte solution. The correlation between 1/m* and the retention times in HIC and RPC was poor due to interaction effects with the chromatography matrices and probably partial unfolding of the proteins; however this parameter (1/m*) was expected to be a measure of hydrophobicity of proteins that relates better than the chromatographic data to experiments where the hydrophobic behavior of proteins in an aqueous solution is used for their separation. The partition. behavior of the five proteins in aqueous two-phase systems (ATPS) in the absence and presence of NaCl was investigated. A poor correlation was found between log K (K is the partition coefficient) in ATPS and the hydrophobicity values measured by RPC and HIC; however a very good correlation was found between log 1/m* which is a measure of protein hydrophobicity based on the solubility of the protein during precipitation and log K, particularly in PEG/PO4 systems with added NaCl. The parameter (1/m*) also demonstrated a good correlation with log K in PEG/dextran systems. A simple correlation for the prediction of partitioning in specific ATPS based on this parameter has been evaluated. An expression describing its resolution power R, and a parameter describing the hydrophobicity of the system, P-o, was determined making the correlation potentially predictive for other proteins in the ATPSs used. Hydrophobicity of proteins was better exploited in PEG/PO4 systems than in PEG/dextran ones as a much higher resolution (R) is obtained in the former.