Some physiological factors which control the rate of induction of the Ca²⁺-induced membrane transition (the term “Ca²⁺-induced membrane transition” is defined in the introduction) have been systematically investigated. To exclude the complicating factors of electron flow and energization, the transition was studied in mitochondria depleted of endogenous substrate, in the presence of uncoupler. In these mitochondria the transition could be induced by Ca²⁺, whether entry was mediated by ruthenium red-sensitive permeation or by A23187 facilitated diffusion. The rate of the transition was reduced fivefold by any agent which caused complete reduction of endogenous NAD. The rate of the transition was increased threefold by the exchange of endogenous ADP for phosphoenolpyruvate. A further increase was found on the addition of atractyloside, but bongkrekic acid caused inhibition. Addition of uncoupler to energized mitochondria when the endogenous NAD was already fully oxidized caused a stimulation of the transition. From these observations we conclude that mitochondria have a set of protective mechanisms (the term “protective mechanism” refers to the means by which these agents inhibit the Ca²⁺-induced transition; such a mechanism could be through allosteric interactions between the sites of binding of inhibitor and Ca²⁺; it would, however, be premature to conclude this on the basis of this paper) involving endogenous NADH, ADP, and energization which regulate the rate of the Ca²⁺-induced transition. ADP appears to work at two sites: one site which is internal, and another at the ADP/ATP translocase. In addition, we conclude that the transition requires neither electron flow nor energy, but rather the mere accessibility of some internal site to Ca²⁺. Finally, the key roles played by the protective agents in metabolism give the cell great potential flexibility in regulating the Ca²⁺-induced transition. This degree of control suggests that the transition has substantial physiological significance.