F-ATP synthases convert the electrochemical energy of the H gradient into the chemical energy of ATP with remarkable efficiency. Mitochondrial F-ATP synthases can also undergo a Ca-dependent transformation to form channels with properties matching those of the permeability transition pore (PTP), a key player in cell death. The Ca binding site and the mechanism(s) through which Ca can transform the energy-conserving enzyme into a dissipative structure promoting cell death remain unknown. Through , and studies we (i) pinpoint the "Ca-trigger site" of the PTP to the catalytic site of the F-ATP synthase β subunit and (ii) define a conformational change that propagates from the catalytic site through OSCP and the lateral stalk to the inner membrane. T163S mutants of the β subunit, which show a selective decrease in Ca-ATP hydrolysis, confer resistance to Ca-induced, PTP-dependent death in cells and developing zebrafish embryos. These findings are a major advance in the molecular definition of the transition of F-ATP synthase to a channel and of its role in cell death.