Mice defective in both granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) have severely impaired neutrophil production and function, yet these mice respond to acute pathogen challenge with a significant neutrophil response. We have recently reported the development of an in vitro system to detect granulopoietic cytokines secreted from cells isolated from G-CSF, GM-CSF double knockout mice. The conditioned media produced by these cells after stimulation with lipopolysaccharide or Candida albicans supports the production and differentiation of granulocytes (ie, the conditioned media contains neutrophil promoting activity [NPA]). We now show that the NPA in the G-CSF(-/-)/GM-CSF(-/-) conditioned media requires interleukin-6 (IL6), is abolished by soluble gp130, and can be specifically immunodepleted by an anti-IL6R antibody. NPA effects on bone marrow cells are also mimicked by Hyper-IL6, and the soluble IL6R is present in NPA. These results show that the IL6/sIL6R complex is the major effector of NPA. NPA production by mice defective for both G-CSF and GM-CSF uncovers an alternative pathway to granulocyte production, which is activated after exposure to pathogens.