A method of separating neutrophils from the peripheral blood of rats with 95% purity is described. To determine the role of antigenic stimulation in neutrophil function, neutrophils from germ-free (GF) rats were compared with those of conventional (CV) rats. Neutrophil counts were lower in GF rats but the total number of monocytic cells was the same. To measure phagocytic killing, superoxide anion production was determined and found to be lower in GF neutrophils (2.1 +/- 0.5 nmol/min/10(6) cells) than in CV neutrophils (9.5 +/- 2.9 nmol/min/10(6) cells). Myeloperoxidase activity was found to be twofold higher in GF neutrophils. When a recombinant human granulocyte colony-stimulating factor (rhG-CSF) was intravenously injected, superoxide production did not change in either GF or CV neutrophils, but the myeloperoxidase activity of neutrophils in both types of rats decreased. rhG-CSF increased the number of neutrophils in both GF (10-fold) and CV rats (three- to fourfold).