The marginal zone is a unique compartment that is only found in the spleen. Rat marginal zone B cells (MZ-B) can be distinguished from other B cells, e.g. recirculating follicular B cells (RF-B), by several phenotypic characteristics. Typically MZ-B cells are surface (s)IgMhi, sIgDlo and CD45R(B220)lo, whereas RF-B cells are sIgMlo, sIgDhi and CD45Rhi. In addition, MZ-B cells stain strongly with HIS57, a newly developed monoclonal antibody. The developmental pathway and origin of MZ-B cells are not exactly known. However, previous studies indicate that recirculating (i. e. thoracic duct) B cells can give rise to MZ-B cells. Here the origin of (naive) MZ-B cells was studied using adriamycin (doxorubicin)-induced B cell depletion. Using three-color flow cytometry and immunohistology we show that 2 days after a single i.v. injection of the anti-tumor drug adriamycin only RF-B cells can be detected, while all other B cell subpopulations are depleted, including all bone marrow precursor B cells. By studying the sequential reappearance of various B cell subsets and their precursors after adriamycin administration we show that MZ-B cells and the splenic marginal zone can be detected at a time point at which newly generated B cells (immature B cells) are not yet present. Given the observation that only RF-B cells were present at this time, we conclude that RF-B cells are the immediate MZ-B precursor cells.