Each of two histidine residues and one tryptophan residue in thermophilic bacterium PS-3 inorganic pyrophosphatase (PPase) was replaced by alanine. The activities of the H125A and W143A variants decreased to one-fifth, whereas the activity of H118A remained unaltered. CD spectra in the near-UV region indicated that the conformations of the first two variants changed with the substitution. In contrast with wild-type PPase, which is hexameric beyond an enzyme concentration of 0.1 microM in the presence of Mg2+, the H118A and H125A variants cannot be assembled from trimers into hexamers at less than an enzyme concentration of 10 microM even at a higher concentration of Mg2+. In particular, H118A was irreversibly inactivated in a diluted state. In contrast, the enzyme concentration dependence of W143A PPase activity was almost the same as that of wild-type PPase. These results indicated that His-118 and His-125 are important for both trimer-trimer interaction and structural integrity, whereas Trp-143 is important structurally. The trimer-trimer interaction is absolutely necessary for the thermostability of the PS-3 enzyme.