MNEI (monocyte/neutrophil elastase inhibitor) is a 42 kDa serpin superfamily protein characterized initially as a fast-acting inhibitor of neutrophil elastase. Here we show that MNEI has a broader specificity, efficiently inhibiting proteases with elastase- and chymotrypsin-like specificities. Reaction of MNEI with neutrophil proteinase-3, an elastase-like protease, and porcine pancreatic elastase demonstrated rapid inhibition rate constants >10⁷ M^-1 s^-1, similar to that observed for neutrophil elastase. Reactions of MNEI with chymotrypsin-like proteases were also rapid:  cathepsin G from neutrophils (>10⁶ M^-1 s^-1), mast cell chymase (>10⁵ M^-1 s^-1), chymotrypsin (>10⁶ M^-1 s^-1), and prostate-specific antigen (PSA), which had the slowest rate constant at ∼10⁴ M^-1 s^-1. Inhibition of trypsin-like (plasmin, granzyme A, and thrombin) and caspase-like (granzyme B) serine proteases was not observed or highly inefficient (trypsin), nor was inhibition of proteases from the cysteine (caspase-1 and caspase-3) and metalloprotease (macrophage elastase, MMP-12) families. The stoichiometry of inhibition for all inhibitory reactions was near 1, and inhibitory complexes were resistant to dissociation by SDS, further indicating the specificity of MNEI for elastase- and chymotrypsin-like proteases. Determination of the reactive site of MNEI by N-terminal sequencing and mass analysis of reaction products identified two reactive sites, each with a different specificity. Cys³⁴⁴, which corresponds to Met³⁵⁸, the P₁ site of α1-antitrypsin, was the inhibitory site for elastase-like proteases and PSA, while the preceding residue, Phe³⁴³, was the inhibitory site for chymotrypsin-like proteases. This study demonstrates that MNEI has two functional reactive sites corresponding to the predicted P₁ and P₂ positions of the reactive center loop. The data suggest that MNEI plays a regulatory role at extravascular sites to limit inflammatory damage due to proteases of cellular origin.