Two-color flow cytometrical (FCM) analysis of rat peripheral lymphoid organs shows two distinct IgM/IgD-defined B cell subpopulations, similar to those of the mouse: a major population of cells expressing little IgM and high levels of IgD (population I) and a minor population of cells expressing high levels of IgM but little IgD (population III). In peripheral lymphoid organs population III cells are mainly found in spleen where they represent about 25% of the B cells; population III cells are almost absent from lymph nodes and Peyer's patches. In adult bone marrow and in neonatal spleen the majority of IgM/IgD-defined B cells (greater than 70%) are population III cells, similar to what is observed in the mouse. In contrast with mice, only a low proportion of the cells (1%) recovered from the peritoneal cavity are B cells, and most of them belong to population I. Previously defined monoclonal antibodies (HIS22 and HIS24) to B cell forms of the leukocyte common antigen (CD45R) in combination with staining for surface IgM and surface IgD demonstrates a further heterogeneity of rat B cells by three-color FCM analyses. HIS22 labels most population I cells; population III cells and a small subset (about one third) of population I express only very low levels of the HIS22 determinant. HIS24 reacts with population I cells and subdivides population III into two subsets: about one third of splenic population III cells are brightly stained with this antibody whereas fluorescence of the remaining two-thirds is lower. The HIS24bright population III cells likely are newly formed B cells since cells with this phenotype are the predominant surface Ig population found in adult bone marrow and neonatal spleen. In tissue sections of lymphoid organs, HIS22- and HIS24-positive cells are mainly found in lymphoid follicles; splenic marginal zones are almost unstained. Combining immunohistological analysis with the FCM data, we therefore conclude that the small follicular B cells are in population I and marginal zone B cells are found in the HIS24dull population III. The in situ localization of HIS24bright population III cells and the HIS22dull population I cells is not clear.(ABSTRACT TRUNCATED AT 400 WORDS)