Earlier we described the synthesis of a toxic, disulfide-linked conjugate between Fragment A from diphtheria toxin (DTA) and the lectin concanavalin A (Con A). Here we report further characterization of this Con A-SS-DTA conjugate and of a nontoxic conjugate containing DTA linked through a disulfide bridge to the binding subunit of ricin toxin, RTB. The Con A-SS-DTA conjugate preparation was heterogeneous, and contained both divalent and tetravalent forms of Con A linked to DTA. The divalent form of the conjugate, which lacked hemagglutination activity, was as toxic as the tetravalent form for 3T3 cells. Consistent with this result, Con A-SS-DTA analogs prepared with the chemically modified dimeric derivatives of Con A, succinyl-Con A and acetyl-Con A, were as toxic as DTA disulfide linked to tetravalent Con A. Two analogs of Con A-SS-DTA containing nonreducible intermolecular linkages between DTA and Con A were at least 1000-fold less toxic than Con A-SS-DTA, although they were indistinguishable from unmodified Con A in binding to 3T3 cells. This suggests that reduction of the disulfide bridge between Con A and DTA may be necessary for conjugate toxicity. Con A-SS-DTA was equally toxic for a variety of cell lines varying in sensitivity to diphtheria toxin, including Vero, CHO, HeLa, and 3T3. The conjugate was also toxic for a diphtheria toxin-resistant variant of V79 cells (V79 Dtxr-3) that has lower affinity than wild type cells for diphtheria toxin. Chloroquine, colchicine, cytochalasin B, and ammonium chloride had no effect on Con A-SS-DTA toxicity for Vero cells, although ammonium chloride and chloroquine inhibited diphtheria toxin action.