The post-translationally acylated Repeats in ToXins (RTX) leukotoxins, such as the adenylate cyclase (CyaA) or α-hemolysin (HlyA), bind β2-integrins of leukocytes, but also penetrate cells lacking these receptors. We show that the indole of conserved tryptophans within the acylated segments, e.g. W876 in CyaA and W579 in HlyA, is crucial for β2-integrin-independent membrane penetration of toxins. Substitutions of W876 by aliphatic or aromatic residues did not affect acylation, folding or activities of CyaA W876L/F/Y toxin variants on CR3-expressing cells. In contrast, toxin activity of CyaA W876L/F/Y on cells lacking CR3 was strongly impaired. Similarly, a W579L substitution selectively reduced HlyA W579L cytotoxicity towards cells lacking β2-integrins. Intriguingly, the W876L/F/Y substitutions increased the thermal stability (Tm) of CyaA by 4 to 8 °C. In addition, the hydrogen-deuterium exchange revealed structural changes in the acylated segment of the CyaA W876F variant, compared to intact CyaA. The substitution of W876 with a polar glutamine (W876Q), not increasing the Tm, or combining of the W876F substitution with a cavity-filling V822M substitution, decreasing the Tm of CyaA W876F+V822M to a value closer to that of CyaA, yielded a milder defect of toxin activity on erythrocytes lacking CR3. Furthermore, the activity of CyaA was selectively impaired on erythrocytes when the interaction of the pyrrolidine of P848 with the indole of W876 was ablated. These results suggest that the bulky indole of the W876 of CyaA, or W579 of HlyA, rules the local structural flexibility of the acylated segment. This may facilitate adoption of a membrane-penetrating conformation of the toxins in the absence of β2-integrin-mediated positioning of the toxin towards the cell membrane.