Neutrophil elastase (NE) and cathepsin G (CG), the proteolytic enzymes localized in azurophil granules of neutrophils (PMN), are involved in PMN responses to various stimuli. When released at sites of inflammation, they participate in the degradation of numerous proteins involved in the regulation of the immune response. In this study, we employed ADAM17-/- fibroblasts stably transfected with cDNA of human pro-tumor necrosis factor α (proTNFα) (ADAM17-/-TNF+) to investigate the effects of NE and CG on shedding and degradation of TNFα. Both NE and CG were found to diminish the level of membrane TNFα (mTNFα) as measured by flow cytometry. This process was accompanied by the accumulation of biologically active soluble TNFα (sTNFα) in the culture medium, as determined by an increase in both the cytotoxic activity of TNFα and its ability to serve as a co-stimulator in the induction of inducible nitric oxide synthase (iNOS). However, in contrast to CG, NE at high concentrations was able to degrade sTNFα released from the cell surface. Using soluble recombinant human TNFα, we identified Val93-Ala94 and Val117-Glu118 as the NE cleavage sites within the sTNFα molecule. Taken together, the ability of NE and CG to modulate levels of membrane and soluble forms of TNFα may contribute to the proinflammatory activity of neutrophils.