Paired immunofluorescent staining with antibodies specific for the major isotypes of mouse immunoglobulin was used to study the ontogenetic expression of diversity of cell surface immunoglobulin. The first B lymphocytes to emerge, derived from cytoplasmic IgM + precursors, express sIgM exclusively. Between birth and 3 days of age separate populations of sIgM + B lymphocytes acquire a second isotype: sIgD, one of the subclasses of sIgG, or sIgA. At 3 days, all splenic B lymphocytes that bear sIgG or sIgA also express sIgM, but virtually none stain for sIgD. By 7 days, a substantial proportion of sIgG + or sIgA + lymphocytes in spleen and most of those in lymph node express both sIgM and sIgD. Anti-μ antibody treatment from birth prevented development of B lymphocytes expressing any isotype. These observations suggest that the immature sIgM + B lymphocyte is the pivotal cell in the generation of the different sublines of B cells and that sIgD is acquired as a third isotype on B lymphocytes already committed to eventual synthesis of IgG or IgA. The frequency of lymphocytes bearing only sIgG or sIgA is higher in old than in young mice, suggesting that sIgD and sIgM may be lost after stimulation by antigens. The occurrence of a nearly identical distribution of sIg isotypes on B lymphocytes from athymic, pathogen-free mice suggests that primary expression of isotype diversity does not require T cells.