Background Granulocyte transfusion has been proposed as a bridging therapy for patients with prolonged periods of chemotherapy-induced neutropenia, who suffer from severe fungal and bacterial infections. To recover, adequate numbers of granulocytes are required when the patients are refractory to standard treatment. The aim of this study was to assess the functional characteristics and efficacy of granulocyte colony-stimulating factor/dexamethasone-mobilized granulocytes used for transfusions. Design and Methods Granulocytes from the leukapheresis products were tested for the expression of cell-surface antigens, interactions with endothelial cells, motility, killing of microbes and survival. The granulocytes were used in vivo for transfusion in 16 severely ill children, who were - apart from a patient with a granulocyte dysfunction - all suffering from prolonged neutropenia. Results Mobilization of granulocytes with granulocyte colony-stimulating factor and dexamethasone caused phenotypic changes (decreased CD62L expression and increased levels of CD66b and CD177). The ability of the granulocytes to interact with endothelial cells (rolling, adhesion, transmigration) and to kill various types of pathogens was not affected by the mobilization, leukapheresis and irradiation procedures. The granulocytes were functionally indistinguishable from those isolated from untreated donors, even after 24 hours of storage. Granulocyte transfusion seemed to benefit 70% of patients, as 11 out of the 16 children showed clinical recovery within 1-2 weeks after beginning the transfusions. Conclusions Although CD62L expression is downregulated on granulocytes used for granulocyte transfusions, concomitant CD177 upregulation may explain the intact interactions with endothelial cells. All other granulocyte functions tested were intact, including the ability to kill fungi. Granulocyte concentrates can be stored without loss of in vitro viability and functionality for at least 24 hours. As demonstrated in vivo, granulocyte transfusions may be an effective therapy for neutropenic pediatric patients suffering from life-threatening infections.