The properties of anion-sensitive ATPase of rat heart mitochondria were studied. Na2CO3, NaHCO3 and Na2SO3 stimualted ATPase activity by 69, 41 and 110%, respectively. Azide, tiocinate and perchlorate inhibited bicarbonate-stimulated ATPase. Bivalent cations increased ATPase activity in such a sequence: Zn2+ greater than or equal to Cd2+ greater than or equal to Co2+ greater than or equal to Mg2+ greater than or equal to Mn2+ greater than Ni2+. In the presence of bicarbonate and sulfite. ATPase activity was maximally stimulated with magnesium. Ni2+ and Ca2+-ions inhibited Mg2+-dependent activity of bicarbonate-stimulated ATPase. AMP uninhibited ATPase activity. The 4 mM concentration of ADP inhibited activity of HCO-3-ATPase. Activity of ATPases decreased at lower temperatures. The properties of anion-sensitive ATPase of rat heart mitochondria and that of HCO-2-ATPase of other cells are discussed.