The major limitation to the use of immunotoxins in the clinic is the toxicity associated with the toxin moiety. BD1-G28-5 single-chain Fv (sFv) is a single-chain immunotoxin targeted to human CD40 and consists of bryodin 1 (BD1), a plant ribosome-inactivating protein that is 20-30-fold less toxic in animals than commonly used toxins, fused to the sFv region of the anti-CD40 monoclonal antibody G28-5. This immunotoxin was expressed in Escherichia coli and purified from refolded inclusion bodies. BD1-G28-5 sFv retained the full protein synthesis inhibition activity of recombinant BD1 and specifically bound to CD40 with a binding affinity, kd, of 1.5 nM, within 10-fold of the bivalent parental monoclonal antibody. BD1-G28-5 sFv was potently cytotoxic against CD40-expressing B lineage non-Hodgkin's lymphoma and multiple myeloma cell lines, with EC50 values in the ng/ml range, but not against a CD40-negative T cell line. Interestingly, BD1-G28-5 sFv was not cytotoxic against CD40-expressing carcinoma cell lines that were sensitive to a BD1-based immunotoxin conjugate targeted to the Ley carbohydrate antigen. These data represent the first report indicating that BD1 can be used in the construction of potent single-chain immunotoxins. Additionally, although BD1-G28-5 sFv effectively killed CD40-expressing hematologic malignancies, its lack of activity against CD40-expressing carcinomas suggests that CD40-mediated trafficking of BD1 differs in the two cancer types.