The adenylate cyclase toxin-hemolysin (CyaA) belongs to the Repeats in ToXin (RTX) family of leukotoxins and plays a key role in the virulence of the whooping cough agent Bordetella pertussis. CyaA translocates its N-terminal adenylyl cyclase (AC) enzyme domain into phagocytes expressing the complement receptor 3 (CR3 aka the αMβ2 integrin CD11b/CD18, or Mac-1), where the AC toxin subverts the bactericidal functions of phagocytes by unregulated conversion of cytosolic ATP to cAMP. In parallel, CyaA permeabilizes cellular membrane by forming small cation-selective pores. With a reduced efficacy, CyaA can also ind and penetrate a variety of epithelial and other host cell types that lack CR3, or even the naked lipid bilayer membranes. Both cytotoxic activities depend on the activation of the protoxin proCyaA to CyaA by covalent fatty acyl modification of two internal lysine residues, Lys860 and Lys983 by a dedicated acyltransferase CyaC. By combining site-directed mutagenesis, CD spectroscopy, mass spectrometry, hydrogen/deuterium exchange, and toxin activity assays, we have identified key residues of the acylated segment of CyaA that are involved in the folding and cell penetration capacity of the CyaA toxin. We hypothesize that the acylated segment plays a key role in the initial anchoring the toxin to the outer leaflet of the lipid bilayer of the plasma membrane of target cells.