Shigella flexneri secretes an enterotoxic, SPATE family autotransporter (AT), SigA, which has cytopathic activity towards cultured epithelial cells. Its cytopathic activity is due to its ability to degrade the cytoskeletal protein, alpha-fodrin. The mechanisms by which AT toxins target cells and tissues differ and the details of how SigA acts are not known. In the current study, the determinants of proteolysis and cell-targeting for SigA were determined. We demonstrate that the SigA passenger or alpha-domain consists of two functionally distinct domains, designated alpha1 and alpha2, which are sufficient to specify proteolytic and cell-binding activities, respectively.