Studies were performed to detect and isolate trace contaminants of staphylococcal enterotoxin B (SEB) in various protein A preparations isolated by affinity chromatography employing human IgG covalently bound to Sepharose 4B. Utilizing an ELISA technique, trace amounts (0.018-0.138%) of SEB could be detected in protein A preparations after separation of the SEB employing a molecular sizing column in a high pressure liquid chromatography (HPLC) system. Trace contamination by SEB could be removed from protein A preparations by an additional DEAE ion exchange chromatography step employing a low ionic strength buffer system (0.005 M NaCl in 0.01 phosphate buffer, pH 7.50). The resulting protein A preparations possessed a purity higher than that observed prior to the final purification step. Polyacrylamide gel electrophoresis (PAGE) analyses of the trace contamination removed from protein A preparations by ion exchange chromatography revealed, in addition to SEB, several additional contaminating polypeptides of an unknown nature. These studies indicate that protein A preparations of high purity can be prepared by employing DEAE ion exchange chromatography in addition to affinity chromatography utilizing immobilized human IgG.