myo-Inositol monophosphatase (myo-inositol-1-phosphate phosphohydrolase, EC 126.96.36.199) is an attractive target for mechanistic investigation due to its critical role in the phosphatidylinositol signaling pathway and the possible relevance of its inhibition by Li+ to manic depression therapy. The x-ray crystallographic structure of human inositol monophosphatase in the presence of the inhibitory metal Gd3+ showed only one metal bound per active site, whereas in the presence of Mn2+, three ions were present with one being displaced upon phosphate binding. We report here modeling, kinetic, and mutagenesis studies on the enzyme, which reveal the requirement for two metal ions in the catalytic mechanism. Activity titration curves with Zn2+ or Mn2+ in the presence or absence of Mg2+ are consistent with a two-metal mechanism. Modeling studies based on the various x-ray crystallographic structures (including those with Gd3+ and substrate bound) further support a two-metal mechanism and define the positions of the two metal ions relative to substrate. While the first metal ion may activate water for nucleophilic attack, a second metal ion, coordinated by three aspartate residues, appears to act as a Lewis acid, stabilizing the leaving inositol oxyanion. In this model, the 6-OH group of substrate acts as a ligand for this second metal ion, consistent with the reduced catalytic activity observed with substrate analogues lacking the 6-OH. Evidence from Tb3+ fluorescence quenching and the two-metal kinetic titration curves suggests that Li+ binds at the site of this second metal ion.