A previous communication from this laboratory as well as one from another described the separation of alpha 2-macroglobulin from swine serum. The products from both laboratories contained, in addition to alpha 2-macroglobulin, an additional macroglobulin contaminant with alpha 2-globulin mobility. Due to their physicochemical similarity these macroglobulins are not resolved using conventional column procedures such as ion exchange chromatography and gel filtration. Subsequent experiments have shown that immunoelectrophoretically pure swine alpha 2-macroglobulin is present, in good yield (65%) in the breakthrough effluent of columns of Bio-Gel A-1.5m-Reactive Blue 2 while the contaminating macroglobulin is tightly bound. The production of highly purified swine alpha 2-macroglobulin utilizing this observation is the subject of the present report. The product of the separation was found to be homogeneous when subjected to immunoelectrophoresis, at a concentration of 14-16 mg/ml, and diffused against antiswine whole serum antibody. The production of monospecific antibody, a more stringent test for homogeneity, resulted when the purified alpha 2-macroglobulin was injected into rabbits. Physicochemical analyses on the purified product showed that swine and human alpha 2-macroglobulins are true homologs.