The reactivity of the mitochondrial (pI = 5) isoenzyme of horse liver aldehyde dehydrogenase was determined by studying the effects of pH on steady-state velocity, burst magnitude, and molecular weight of the enzyme in the absence and presence of Mg2+ ions. The Mg2+ ion activation of the steady-state velocity at pH 7.5 has been explained through a mechanism involving alteration of the tetrameric enzyme, functioning with half-of-the-sites reactivity, to a dimeric enzyme, functioning with all-of-the-sites reactivity [Takahashi, K., & Weiner, H. (1980) J. Biol. Chem. 255, 8206-8209]. With increasing pH, the tetrameric enzyme dissociated even in the absence of Mg2+ ions to the more active dimeric state. The pH-dependent dissociation was governed by proton release from a group with pK = 9.5. After correcting for the increased number of functioning active sites, determined from the pre-steady-state burst, we calculated that elevated pH also caused an increase in the velocity of the rate-limiting step, hydrolysis of the acyl-enzyme intermediate. This event was governed by the ionization of two groups, with pK = 7.2 and 9.5, respectively. If these groups are directly involved in the catalytic step, a mechanism involving histidine acting as a general base can be proposed for the former group. The latter group may be involved in a charge relay activation process which only occurs at elevated, nonphysiological pH. The importance of the latter is questionable, as there is only a 3-fold increase in Vmax when this group is involved in catalysis.