The effects of Mg2+ and Ca2+ ions on the activities of mitochondrial and cytosolic aldehyde dehydrogenases isolated from horse, rat, and beef livers were investigated in 0.1 M sodium phosphate, pH 7.5, at 25 degrees C. As with the Mg2+-enhancement of the horse liver mitochondrial enzyme [17], Mg2+-activation was observed for the mitochondrial enzymes from rat and beef. The cytosolic enzymes from horse, rat, and beef livers were inhibited in the presence of Mg2+ ions. The effects of Ca2+ ions on the activity were essentially the same as those observed in the presence of Mg2+ ions; the mitochondrial isozymes were activated while the cytosolic isozymes were inhibited. The fact that only the activity of mitochondrial forms of mammalian liver aldehyde dehydrogenase was enhanced by Ca2+ or Mg2+ ions may be related to the in vivo regulation of aldehyde metabolism, a presumed mitochondrial event.