Cytotoxic necrotizing factor 1 (CNF1) is a protein toxin produced by pathogenic Escherichia coli strains. CNF1 constitutively activates small GTPases of the Rho family by deamidation of a glutamine, which is crucial for GTP hydrolysis. The toxin is taken up into mammalian cells by receptor-mediated endocytosis and is delivered from late endosomes into the cytosol. Here, we show that an approximately 55-kDa fragment of CNF1, which contains the catalytic domain and an additional part of the toxin, is present in the cytosol. The processing of this fragment requires an acidic pH and insertion of the toxin into the endosomal membrane. We define the cleavage site region as the region located between amino acids 532 and 544 of CNF1. The data provide insight into the complex mechanism of uptake of bacterial toxins into mammalian cells.