Although the capacity of isolated β-subunits of the ATP synthase/ATPase to perform catalysis has been extensively studied, the results have not conclusively shown that the subunits are catalytically active. Since soluble F1 of mitochondrial H+-ATPase can bind inorganic pyrophosphate (PPi) and synthesize PPi from medium phosphate, we examined if purified His-tagged β-subunits from Thermophilic bacillus PS3 can hydrolyze PPi. The difference spectra in the near UV CD of β-subunits with and without PPi show that PPi binds to the subunits. Other studies show that β-subunits hydrolyze [32P] PPi through a Mg2+-dependent process with an optimal pH of 8.3. Free Mg2+ is required for maximal hydrolytic rates. The Km for PPi is 75 μM and the Vmax is 800 pmol/min/mg. ATP is a weak inhibitor of the reaction, it diminishes the Vmax and increases the Km for PPi. Thus, isolated β-subunits are catalytically competent with PPi as substrate; apparently, the assembly of β-subunits into the ATPase complex changes substrate specificity, and leads to an increase in catalytic rates.