Phagocytosis and intracellular destruction of pathogens by phagocytes is a crucial defense mechanism of the innate immune response during infection. It has been reported a number of times that the interaction with pyogenic, extracellular bacteria leads to the apoptotic death of phagocytes. The signaling events that cause this form of cell death are largely unknown. In this study, we demonstrate a link between uptake, killing and degradation of Escherichia coli bacteria and induction of apoptosis in macrophages. Treatment of murine RAW 264.7 macrophages with bafilomycin A(1), a phagosome acidification inhibitor, reduced killing and degradation of phagocytosed bacteria and significantly decreased macrophage apoptosis. The stable overexpression of constitutively active or dominant-negative mutants of the small GTPase Rab5a increased bacterial phagocytosis and consecutively apoptosis. In these cells, relative killing and degradation were not affected, linking the increased apoptosis to enhanced uptake and suggesting that the apoptosis-inducing signal derives from the higher incidence of degradation events or an accumulation of phagosomes of a late maturation stage. These results thus provide a link between bacterial phagocytosis and degradation and the induction of apoptosis in macrophages. We propose that this form of apoptosis is the physiological conclusion of an innate immune response against pyogenic bacteria.