Electrospray ionization mass spectrometry (ESI-MS) was used to study the noncovalent metallo-enzynae-inlaibitor complexes of matrilvsin (a matrix metalloproteinase of mass 18,720 u) under gentle experimental conditions" and to determine the metal ion association stoichiometries in both the free enzyme and the complexes. The metal association stoichiometries of the free matrilvsin were found to be highly sensitive to solution pH changes. At pH 2.2 the enzvrne existed as metal-free apo-matrilysin and was not capable of binding an inhibitor. Ai pH 4.5-7.0 the enzyme associated specifically with zinc and calcium cations and became active in inhibitor binding. Although the stoichiometries of the metal cofactors varied (zero to two zinc and/or calcium ions) in the free enzyme dependent on solution pH, the predominant form of the enzyme-inhibitor complexes in the pH range of 4.5-7.0, in contrast, always had the metal assoc'iation stoichiometry of 2Zn + 2Ca, which was the same stoichiometry the most active free metallo-enzyme had at the optimal pH of 7. At the activity onset pH of 4.5 matrilvsin existed mostly as ap0-enzyme (but in a conformation different from the denatt, red one at pH 2.2) and 1.4ound to an inhibitor slowly (time constant ~ 2.5 rain) to form the noncovalent mctallo-enzyme-inhibitor complex. Of the two inhibitors studied, the one with the higher solution binding constant also produced larger ion signals for the noncovalent complex in the solvent-free gas phase, which pointed to the feasibility of the use of ESI-MS for inhibitor screening studies.