TRAIL induces apoptosis in a variety of tumor cells. Our laboratory found that human neutrophils contain an intracellular reservoir of prefabricated TRAIL that is released after stimulation with Mycobacterium bovis bacillus Calmette-Guérin. In this study, we examined the subcellular distribution of TRAIL in freshly isolated neutrophils. Neutrophil granules, secretory vesicles (SV), and plasma membrane vesicles were isolated by subcellular fractionation, followed by free-flow electrophoresis, and examined by ELISA and immunoblot. TRAIL was found in all membrane-bound fractions with the highest amounts in the fractions enriched in azurophilic granule (AG) and SV. Immunofluorescence confocal microscopy showed that TRAIL colocalized independently with myeloperoxidase (MPO), lactoferrin (LF), and albumin, respective markers of AG, specific granules, and SV. Furthermore, immunotransmission electron microscopy demonstrated that TRAIL colocalized intracellularly with MPO and albumin. We examined TRAIL expression in PLB-985 cells induced with dimethylformamide and in CD34-positive stem cells treated with G-CSF. Quantitative RT-PCR analysis showed that TRAIL was expressed in each stage of development, whereas MPO and LF were only expressed at distinct times during differentiation. Collectively, these findings suggest that TRAIL is expressed throughout neutrophil development, resulting in a broad distribution among different granule subtypes.