Shiga toxin can be internalized by clathrin-dependent endocytosis in different cell lines, although it binds specifically to the glycosphingolipid Gb3. It has been demonstrated previously that the toxin can induce recruitment of the toxin-receptor complex to clathrin-coated pits, but whether this process is concentration-dependent or which part of the toxin molecule is involved in this process, have so far been unresolved issues. In this article, we show that the rate of Shiga toxin uptake is dependent on the toxin concentration in several cell lines [HEp-2, HeLa, Vero and baby hamster kidney (BHK)], and that the increased rate observed at higher concentrations is strictly dependent on the presence of the A-subunit of cell surface-bound toxin. Surface-bound B-subunit has no stimulatory effect. Furthermore, this increase in toxin endocytosis is dependent on functional clathrin, as it did not occur in BHK cells after induction of antisense to clathrin heavy chain, thereby blocking clathrin-dependent endocytosis. By immunofluorescence, we show that there is an increased colocalization between Alexa-labeled Shiga toxin and Cy5-labeled transferrin in HeLa cells upon addition of unlabeled toxin. In conclusion, the data indicate that the Shiga toxin A-subunit of cell surface-bound toxin stimulates clathrin-dependent uptake of the toxin. Possible explanations for this phenomenon are discussed.