In vitro studies have defined an essential role for stromal cells in supporting B-cell development, including production of lymphopoietic cytokines. It has been suggested that stromal cells are equivalent to adventitial reticular cells in the marrow; however, evidence of reticular cells producing cytokines has been difficult to obtain. Staining of bone marrow (BM) sections with antibodies to interleukin-7 (IL-7) showed a reticular pattern, mimicking that obtained using antibodies to vascular cell adhesion molecule 1 (VCAM-1), a molecule present on both stromal cells in vitro and reticular cells. To more closely examine cytokine production within normal marrow, an immunomagnetic separation scheme was devised to directly enrich VCAM-1+ stromal cells. Twenty to thirty percent of cells isolated in the VCAM-1+ fraction shared characteristics with stromal cells from long term BM cultures, including cellular morphology and expression of alkaline phosphatase and alpha actin. These were termed “reticular stromal” cells. Immunohistochemical staining showed that virtually all of the latter cells possessed cytoplasmic IL-7 protein, and about half expressed stem cell factor. In contrast with cultured stromal cells, very few had detectable macrophage-colony-stimulating factor. These data constitute the first report of cytokine expression by marrow reticular cells in vivo. The implications of this data with respect to the existence of stromal cell subsets and their regulation of lymphopoiesis is discussed.