Staphylococcus aureus is a leading cause of bacterial infections in humans, including life-threatening diseases such as pneumonia and sepsis. Its small membrane-pore-forming alpha-toxin is considered an important virulence factor. By destroying cell-cell contacts through cleavage of cadherins, the metalloproteinase ADAM10 (a disintegrin and metalloproteinase 10) critically contributes to alpha-toxin-dependent pathology of experimental S. aureus infections in mice. Moreover, ADAM10 was proposed to be a receptor for alpha-toxin. However, it is unclear whether the catalytic activity or specific domains of ADAM10 are involved in mediating binding and/or subsequent cytotoxicity of alpha-toxin. Also, it is not known how alpha-toxin triggers ADAM10's enzymatic activity, and whether ADAM10 is invariably required for all alpha-toxin action on cells. In the present study, we show that efficient cleavage of the ADAM10 substrate epithelial cadherin (E-cadherin) requires supra-cytotoxic concentrations of alpha-toxin, leading to significant increases in intracellular [Ca2+]; the fall in cellular ATP levels, typically following membrane perforation, became observable at far lower concentrations. Surprisingly, ADAM10 was dispensable for alpha-toxin-dependent xenophagic targeting of S. aureus, whereas a role for a-toxin attack on the plasma membrane was confirmed. The catalytic site ofADAM10, furin cleavage site, cysteine switch and intracellular domain of ADAM10 were not required for alpha-toxin binding and subsequent cytotoxicity. In contrast, an essential role for the disintegrin domain and the prodomain emerged. Thus, co-expression of the prodomain with prodomain-deficient ADAM10 reconstituted binding of alpha-toxin and susceptibility of ADAM10-deficient cells. The results of the present study may help to inform structural analyses of alpha-toxin-ADAM10 interactions and to design novel strategies to counteract S. aureus alpha-toxin action.