Chemical modifications with water-soluble carbodiimides (EDC and CMC) were performed to elucidate whether some carboxyl residues are involved in the catalytic activity of membrane-bound pyrophosphatase of Rhodospirillum rubrum. EDC and CMC cause a loss of hydrolytic activity following pseudo-first-order kinetics up to 10 min of reaction. The enzyme was completely protected against EDC inhibition by PPi or Mg2+, whereas PPi or Mg2+ gave partial protection against CMC inactivation. Mg-PPi protected completely against the inhibition caused by both carbodiimides. These data suggest that the carboxyl moiety modified by EDC is at the active site. At longer times of inactivation with both carbodiimides, we could not observe a linear relationship in semilogarithmic plots of residual activity versus time, indicating that at least two carboxyls are involved in the inactivation, which correlates with the partial protection against CMC inactivation by PPi. We found that the activator site for Mg2+ is apparently at or near the active site of the enzyme. This is supported by the fact that PPi protects completely the activator effect of this divalent cation.