Neutrophil apoptosis is delayed under trauma and/or sepsis conditions. The mechanism for the delay has remained unclear. We hypothesize that modulation of the mitochondrial pathway of apoptosis contributes to the delay in neutrophil apoptosis with burn injury. Rats were subjected to burn injury (30% total body surface area, 98°C for 10 sec), and euthanatized 24 hours post-injury. Blood neutrophils from sham and burn injured rats were isolated using ficoll gradient centrifugation, and cultured for 2 or 8 hours. Neutrophil apoptosis was determined using annexin V and propidium iodide (PI) labeling and flow cytometry. Neutrophil mitochondrial morphology was assessed via histochemical staining (MitoTracker GreenFM) and confocal microscopy. Neutrophils from rats with burn injury showed decreased level of apoptosis, compared to sham rat neutrophils at both 2 and 8 hr incubations. In incubated sham rat neutrophils, mitochondria showed a change from normal "tubular" to an "aggregated" morphology. In contrast, cultured neutrophils from burn rats did not exhibit this mitochondrial morphological transition until 8-hr incubation. Compared to sham rat neutrophils, neutrophils from burn rats showed decreased levels of active capspase-9 and -3. Whereas an up-regulation of Bcl-xl and a down-regulation of Bax seemed to contribute to decreased apoptosis in burn rat neutrophils at 2 hr incubation, the decreased apoptosis at 8 hr appeared to be associated with a decrease in Bax and increased phosphorylated Bad. These data suggest that suppression of the mitochondrial pathway plays an essential role in the delay of PMN apoptosis with burn injury.