The cytotoxicity of ricin and diphtheria toxin was studied in culture-adapted bloodstream forms of Trypanosoma brucei. Although ricin is endocytosed at a rate comparable to that of other internalized macromolecules, it is nontoxic to bloodstream-form trypanosomes. The resistance lies partly in low susceptibility of the targeted ribosomes: T. brucei cell-free protein biosynthesis is only partially inhibited by ricin A chain. In addition, ricin is degraded before it reaches the ribosomes, as the toxin is delivered to lysosomes. In contrast, diphtheria toxin shows similar cytotoxicities for bloodstream-form trypanosomes and mouse myeloma cells. Both trypanosome and myeloma cells are more than 1000-fold less sensitive to the action of the toxin than most other mammalian cell lines, although nicked reduced diphtheria toxin inhibits cell-free protein synthesis of T. brucei and myeloma cells to the same extent as that of a rabbit reticulocyte lysate. The effect of diphtheria toxin on T. brucei in vitro translation is NAD+ dependent, suggesting that ADP-ribosylation of elongation factor 2 could be the cause of the inhibition as it is in mammalian cells. Thus, the toxic moiety of diphtheria toxin is suitable for preparation of cell-type-specific cytotoxic reagents directed towards trypanosomes.