A previous study on cleavage of disulfide bonds in endocytosed model compounds had shown that an initial phase of cleavage was totally inhibited by membrane-impermeant sulfhydryl inhibitors and thus was mediated by cell surface sulfhydryls (Feener, E. P., Shen, W.-C., and Ryser, H. J.-P. (1990) J. Biol. Chem. 265, 18780-18785). This paper uses the same inhibitors (5,5'-dithiobis(2-nitrobenzoic acid) and p-chloromercuriphenylsulfonic acid) to examine the role of surface sulfhydryls in the cytotoxicity of diphtheria toxin (DT). Since the interchain disulfide of endocytosed DT must be cleaved prior to translocation of chain A from endosomes to cytoplasm, it was postulated that surface sulfhydryls might mediate the cleavage of that disulfide bond as well. Both sulfhydryl blockers did indeed markedly inhibit DT cytotoxicity. This effect was not due to inactivation of unbound DT, inhibition of receptor-mediated endocytosis, or impairment of acidification of endosomes. We conclude that cell surface sulfhydryls susceptible to blockage by 5,5'-dithiobis(2-nitro-benzoic acid) and p-chloromercuriphenylsulfonic acid are required for the cytotoxicity of DT and, most likely, for the reductive cleavage of DT's interchain disulfides. Ricin cytotoxicity was not decreased; this is consistent with the view that ricin reaches the cytoplasm from a late endocytic structure and with the finding that endocytosed disulfides are also cleaved in a cell fraction containing elements of the Golgi apparatus (Feener, E. P., Shen, W.-C., and Ryser, H. J.-P. (1990) J. Biol. Chem. 265, 18780-18785).