Diphtheria toxin (DT) is a classic A-B toxin; the A chain of the toxin (DTA) contains the enzymatic activity (ADP-ribosyltransferase) and the B chain (DTB) binds to cell surface receptors and initiates internalization of the toxin into the cell. We have mutagenized sites within both DTA and DTB in an attempt to individually inactivate these functions. Mutants with reduced enzymatic activity could be useful as an alternative vaccine while mutants that reduce binding activity yet maintain translocation and enzymatic activity could be useful in the construction of immunotoxins. Two substitutions within the A chain of the toxin (H21G, G79D) were found to greatly reduce the enzymatic activity of the toxin yet did not appear to alter the overall conformation of the toxin. Both mutants displayed similar trypsin digestion patterns and both bound monoclonal antibodies against conformational determinants in a manner similar to that of the wild type toxin. These two DTA substitutions were combined with either wild type DTB or a DTB mutant (S525F) known to have an 8,000-fold reduction in binding. Toxins containing these single, double or triple mutations were expressed in E. coli and purified using nickel affinity chromatography. The potential of these genetically inactivated toxins to produce a neutralizing immune response is currently being examined. Three mutants with substitutions in the B chain of the toxin, S508F, S525F, and F530A are also being examined. These mutations reduce receptor binding but are not believed to alter either the translocation or the enzymatic activity of the toxin. These mutants are currently being combined, both chemically and genetically, with a monoclonal antibody against the human transferrin receptor and tested for their potential as immunotoxins.