Shiga toxin (Six) is internalized by receptor-mediated endocytosis and transported retrogradely to the endoplasmic reticulum from where the enzymatically active part of the toxin is translocated to the cytosol. In this study, we have investigated the effect of polyunsaturated fatty acids (PUFA) on intoxication and retrograde transport of Six. In HEp-2 cells, PUFA treatment inhibited Stx intoxication by a factor of 10. Moreover, both Six internalization and endosome-to-Golgi transport were reduced by PUFA and these reductions can together explain the reduced toxicity. Also cholera toxin internalization was reduced by PUFA treatment. Finally, ricin and Pseudomonas exotoxin I cytotoxicity were not reduced by PUFA, demonstrating that PUFA do not cause a general block in retrograde transport to the endoplasmic reticulum. In conclusion, these results clearly demonstrate the importance of PUFA for Six and cholera toxin trafficking. (C) 2007 Elsevier Inc. All rights reserved.