Inhibition of human chymase by the serpins alpha1-antichymotrypsin (ACT) and alpha1-proteinase inhibitor (PI) at pH 8.0 produces a complex stable to dissociation by SDS/dithiothreitol and a second product, hydrolyzed/inactivated serpin. The first product is the presumed trapped acyl-enzyme complex typical of serpin inhibition, and the second is the result of a concurrent substrate-like reaction. As a result of the hydrolytic reaction, stoichiometries of inhibition (SI) appear greater than 1; values of 4 and 6.0 are observed for the chymase-ACT and -PI reactions. In this study the effect of pH on the inhibition rate constant (kinh) and the SI of each reaction were evaluated to better define the rate-limiting steps of the inhibitory and hydrolytic reaction pathways associated with chymase inhibition. Reactions were evaluated over a pH range to correlate kinh and SI with the ionizations (pK values of 7 and 9) that typically regulate serine protease catalytic activity. The results show that the effects of pH on SI and kinh differ for each inhibitor. On reducing the pH from 8.0 to 5.5, the chymase-ACT reaction exhibited a decrease in SI (to about 1) and little change in kinh, whereas the chymase-PI reaction revealed an increase in SI and a marked decrease in kinh. On increasing the pH from 8.0 to 10.0, the chymase-ACT reaction exhibited little change in SI and a marked decrease in kinh, whereas the chymase-PI reaction revealed a decrease in SI and a marked increase in kinh. Chymase catalytic properties determined for a peptide substrate were atypical over the high pH range exhibiting increases for kcat/Km and kcat and decreases for Km. This behavior suggests the presence of a high pH enzyme form with enhanced hydrolytic activity. From these results and others involving analyses of ACT/PI reactive loop chimeras and ACT point variants exhibiting a range of SI values, we suggest that the diverse pH effects on kinh and SI are caused largely by a difference in the abilities of ACT and PI to interact with low (catalytically inactive) and high (catalytically enhanced) pH forms of chymase. The constancy of kinh for the chymase-ACT reaction over the low pH range suggests that the rate-limiting step for inhibition is pH insensitive and not reflective of diminished chymase hydrolytic activity. Low pH did not appear to affect the rate of SDS-stable complex formation as complex accumulation, assessed qualitatively by SDS-PAGE, correlated with the loss of chymase enzymatic activity.