Neutrophils are short-lived leukocytes that die by apoptosis. Whereas stress-induced apoptosis is mediated by the p38 mitogen-activated protein (MAP) kinase pathway (Frasch, S. C., Nick, J. A., Fadok, V. A., Bratton, D. L., Worthen, G. S., and Henson, P. M. (1998) J. Biol. Chem. 273, 8389-8397), signals regulating spontaneous neutrophil apoptosis have not been fully determined. In this study we found increased activation of protein kinase C (PKC)-beta and -delta in neutrophils undergoing spontaneous apoptosis, but we show that only activation of PKC-delta was directly involved in the induction of apoptosis. PKC-delta can be proteolytically activated by caspase 3. We detected the 40-kDa caspase-generated fragment of PKC-delta in apoptotic neutrophils and showed that the caspase 3 inhibitor Asp-Glu-Val-Asp-fluoromethylketone prevented generation of the 40-kDa PKC-delta fragment and delayed neutrophil apoptosis. In a cell-free system, removal of PKC-delta by immunoprecipitation reduced DNA fragmentation, whereas loss of PKC-alpha, -beta, or -zeta had no significant effect. Rottlerin and LY379196 inhibit PKC-delta and PKC-beta, respectively. Only Rottlerin was able to delay neutrophil apoptosis. Inhibitors of MAP-ERK kinase 1 (PD98059) or p38 MAP kinase (SB202190) had no effect on neutrophil apoptosis, and activation of p42/44 and p38 MAP kinase did not increase in apoptotic neutrophils. We conclude that spontaneous neutrophil apoptosis involves activation of PKC-delta but is MAP kinase-independent.