The exact mechanism of apoptosis in neutrophils (PMNs) and the explanation for the antiapoptotic effect of granulocyte colony-stimulating factor (G-CSF) in PMNs are unclear. Using specific fluorescent mitochondrial staining, immunofluorescent confocal microscopy, Western blotting, and flow cytometry, this study found that PMNs possess an unexpectedly large number of mitochondria, which are involved in apoptosis. Spontaneous PMN apoptosis was associated with translocation of the Bcl-2-like protein Bax to the mitochondria and subsequent caspase-3 activation, but not with changes in the expression of Bax. G-CSF delayed PMN apoptosis and prevented both associated events. These G-CSF effects were inhibited by cycloheximide. The general caspase inhibitor z-Val-Ala-DL-Asp-fluoromethylketone (zVAD-fmk) prevented caspase-3 activation and apoptosis in PMNs, but not Bax redistribution. PMN-derived cytoplasts, which lack a nucleus, granules, and mitochondria, spontaneously underwent caspase-3 activation and apoptosis (phosphatidylserine exposure), without Bax redistribution. zVAD-fmk inhibited both caspase-3 activation and phosphatidylserine exposure in cultured cytoplasts. Yet, G-CSF prevented neither caspase-3 activation nor apoptosis in cytoplasts, confirming the need for protein synthesis in the G-CSF effects. These data demonstrate that (at least) 2 routes regulate PMN apoptosis: one via Bax-to-mitochondria translocation and a second mitochondria-independent pathway, both linked to caspase-3 activation. Moreover, G-CSF exerts its antiapoptotic effect in the first, that is, mitochondria-dependent, route and has no impact on the second.