Staphylococcus aureus secretes a family of potent, noncovalent inhibitory proteins that selectively target the neutrophil serine proteases neutrophil elastase, cathepsin-G, and proteinase-3. A majority of our understanding of these so-called EAP domain proteins has come from structure/function studies on EapH1 and its effects on human neutrophil elastase (hNE). Inspection of the EapH1/hNE cocrystal structure suggested that EapH1 residues R89, E94, and K95 are positioned near the EapH1/hNE interface and might contribute to the potent inhibition of hNE by EapH1. In this study, we used site directed mutagenesis, kinetic evaluation, and surface plasmon resonance to probe the individual contributions of R89, E94, and K95 to EapH1 function. We found that the wild-type EapH1/hNE complex is characterized by a fast association rate (2.0 × 106 M-1 s-1) and a very slow dissociation rate (4.3 × 10-5 s-1), yielding an apparent inhibition constant of 21 pM. The slow dissociation rate of EapH1 from hNE resulted in a time-dependent inhibition pattern. Although conservative mutants E94Q and K95M, as well as the E94Q/K95M double mutant, had on- and off-rates comparable to wild-type EapH1, mutation of R89 to methionine resulted in a 15,000-fold decrease in inhibition (321 nM) and loss of the time-dependent inhibition characteristic. The double mutants R89M/E94Q and R89M/K95M, as well as the triple mutant R89M/E94Q/K95M were similarly perturbed. Mutation of R89 to lysine restored a portion of the inhibition of hNE (27 nM). Given these observations, we conclude that R89 is a primary contributor to EapH1 function vis-à-vis time-dependent inhibition of hNE.