Superoxide anion (O2-) generation by human blood neutrophils induced by phorbol myristate acetate, formyl-methionyl-leucyl-phenylalanine, and monoclonal antibody YI51 was measured 24 h after incubation in medium alone, medium with recombinant human granulocyte colony-stimulating factor (rG-CSF), and medium with lipopolysaccharide (LPS). Monoclonal antibody YI51 was able to bind to neutrophils and induce O2- generation after the addition of anti-mouse immunoglobulin antibody as a cross-linking agent. In the 24-h culture, there was no significant difference in neutrophil survival among the three cultures. The amount of O2- generated by neutrophils in control medium markedly decreased compared with that before culture. However, cells in medium with rG-CSF or LPS maintained the ability to generate O2- well or moderately, respectively. Thus, the activity maintained by rG-CSF and LPS was neutralized by the anti-G-CSF serum. Furthermore, significant amounts of G-CSF were detected in supernatants of neutrophils cultured with LPS for 24 h. It was not detectable, however, in control supernatants. To examine whether the phenotype of the plasma membrane of cells changed in the 24-h culture, expression of CD16 (FcR III) and YI51 antigens was analyzed by flow cytometry. The expression of CD16 and YI51 antigens on cells cultured with rG-CSF or LPS was maintained compared with that of control cells. These observations thus indicate that G-CSF is one of the factors essential to maintain the functioning and phenotype of mature neutrophils.