Conjugates between monoclonal antibodies recognizing human cancer cells and the superantigen staphylococcal enterotoxin A (mAb-SEA) represent a potential novel approach to tumor therapy. Such mAb-SEA conjugates direct T-cells to lyse colon carcinoma cells in vitro. The synthesis of mAb-SEA conjugates which were prepared by introducing thiol groups on SEA and iodoacetyl or maleimide groups on mAb forming a stable thioether linkage between SEA and mAb is described. A hydrophilic spacer, composed of repeated ethylene oxide units, was constructed to increase the distance between SEA and mAb, preserving biological activity of both proteins. The degree of modification of mAb with SEA was determined with SDS-PAGE. Variables influencing the composition of the conjugates and their effect on the tumor-cell cytotoxicity were studied and optimal conditions for the synthesis were established. Functionally active mAb-SEA conjugates were prepared from a panel of different mAb and T-cell-dependent cytotoxicity against several human cancer types including colon, ovarial, breast, and renal cancer was obtained. This suggests that mAb-SEA conjugates may be of value in the treatment of human neoplastic disease.