Serine esterase activity has been implicated in the neutrophil function of lysosomal enzyme release. To better characterize this esterase role and its possible plurality, the effect of synthetic protease inhibitors on complement- and noncomplement-mediated enzyme secretion was investigated. Noncytotoxic, selective degranulation was achieved by exposing human neutrophils to either 10% Zymosan-Activated Plasma (ZAP) or 0.0001M N-formyl-Methionyl-Phenylalanine (fMP) in the presence of 5 microgram/ml of Cytochalasin B. The majority of the releasing capability generated with ZAP was shown to be C5-related using C5-deficient plasma. Lysosomal markers assayed were Beta-Glucuronidase for primary granule response and Lysozyme for secondary granule involvement. The inhibitors were used to pretreat the cells or were added concomitantly with the releasing stimulus. The tryptic inactivator TLCK inhibited both primary and secondary granule release regardless of the stimulus and inactivation protocol. The chymotryptic inhibitor, TPCK, only blocked fMP-induced release in the absence of inducer; however, when present with either stimulus, secondary granule secretion alone was inhibited. The sulfonyl halide, PMSF, did not impede release under any of the experimental conditions. The chymotrypsin model substrate, BTEE, blocked secondary release by both ZAP and fMP only when present with the secretory stimuli. Its counterpart for tryptic activity, TAME, had no effect on release. These studies suggest the involvement of tryptic and chymotryptic activated esterases in lysosomal enzyme release from human neutrophils.