Neutrophil apoptosis plays a central role in the resolution of granulocytic inflammation. We have shown previously that tumor necrosis factor-α (TNFα) enhances the rate of neutrophil apoptosis at early time points via a mechanism involving both TNF receptor (TNFR) I and TNFRII. Here we reveal a marked but consistent variation in the magnitude of the pro-apoptotic effect of TNFα in neutrophils isolated from healthy donors, and we show that inhibition of cell surface aminopeptidase N (APN) using actinonin, bestatin, or inhibitory peptides significantly enhanced the efficacy of TNFα-induced killing. Notably, an inverse correlation is shown to exist between neutrophil APN activity and the sensitivity of donor cells to TNFα-induced apoptosis. Inhibition of cell surface APN appears to interfere with the shedding of TNFRI, and as a consequence results in augmented TNFα-induced apoptosis, cell polarization, and TNFα-primed, formyl-methionyl-leucyl-phenylalanine-stimulated respiratory burst. Of note, actinonin and bestatin had no effect on TNFRII expression under resting or TNFα-stimulated conditions and did not alter CXCRI or CXCRII expression. These data suggest significant variation in the activity of APN/CD13 on the cell surface of neutrophils in normal individuals and reveal a novel mechanism whereby APN/CD13 regulates TNFα-induced apoptosis via inhibition of TNFRI shedding. This has therapeutic relevance for driving neutrophil apoptosis in vivo.