An ATP pyrophosphohydrolase in a rat liver plasma-membrane subfraction was studied with respect to specific Ca2+ activation of the beta-phosphate bond hydrolysis. ATP and, in addition, adenosine 5'-[betagamma-imido]triphosphate and adenosine 5'-[betagamma-methlylene]triphosphate were substrates for Ca2+-stimulated enzymic hydrolysis of the beta-phosphate bond. A 15-fold activation was observed by raising the free Ca2+ concentration from 10(-7) to 10(-5) M. Mg2+ had little effect. Solubilization in 1% deoxycholate and partial purification on a sucrose density gradient resulted in a 5-fold increase in specific activity with unaltered Ca2+-stimulation pattern. The possible importance of the enzyme in Ca2+ transport is discussed.