A two-step method for the isolation of an IgG1 monoclonal antibody against horseradish peroxidase from hybridoma cell culture supernatant is described. Purification was achieved using an aqueous two-phase extraction procedure in conjunction with thiophilic adsorption chromatography. In an aqueous two-phase system composed of 5% PEG 1540 and 22% phosphate the monoclonal antibody preferentially associates with the PEG-rich top phase whereas proteins such as albumin and transferrin partition into the salt-rich bottom phase. Final purification of the monoclonal antibody was achieved by subjecting the PEG-rich top phase to thiophilic adsorption chromatography. The monoclonal antibody purified to homogeneity retained its specificity for horseradish peroxidase as revealed by polyacrylamide gel electrophoresis and an enzyme-linked immunosorbent assay. The potential of this purification protocol for large scale applications is discussed.