IL-6 is a pleiotropic cytokine involved in the regulation of hematopoiesis and lymphocyte activation. An extremely sensitive ELIspot assay was developed to detect and enumerate individual cells secreting IL-6 in normal BALB/c mice. Under physiologic conditions, the frequency of cells producing IL-6 in the bone marrow, spleen, and mesenteric lymph nodes of normal adult animals was approximately 0.5, 0.1, and 0.01%, respectively. FACS analysis revealed that macrophages and Mac1- B220- cells accounted for the vast majority of IL-6 production in the bone marrow, whereas T and B lymphocytes accounted for 4% and 38% of IL-6 production in the spleen. Simultaneous studies comparing cell number with IL-6 levels indicated that the average activated cell produced approximately 85 molecules of IL-6/s.