HeLa cells exposed to a single sublethal concentration of diphtheria toxin were found to have diminished sensitivity when subsequently reexposed to the toxin. Three cells strains exhibiting toxin resistance were developed. In the cells that had previously been exposed to toxin at 0.015 mug/ml, 50% inhibition of protein synthesis required a toxin concentration of 0.3 mug/ml, which is more than 10 times that required in normal HeLa cells. There appears to be a threshold level of diphtheria toxin action. Concentrations of toxin greater than that required for 50% inhibition of protein synthesis (0.01 mug/ml) are associated with cytotoxicity, whereas those below this concentration may not be lethal. Several established human cell lines of both normal and neoplastic origin were tested for their sensitivity to the effects of the toxin. No special sensitivity was observed with the cells of tumor origin. Fifty % inhibition of protein synthesis of HeLa cells was achieved with diphtheria toxin (0.01 mug/ml) as compared to the normal human cell lines tested (0.03 and 0.5 mug/ml) and a cell line derived from a human pancreatic adenocarcinoma (0.2 mug/ml). A human breast carcinoma cell line showed a maximum of 45% inhibition of protein synthesis. This required a diphtheria toxin concentration of 5 mug/ml. These results suggest that different human cell lines show wide variation in their sensitivity to the toxin.