This study describes the purification of a subset of tonsillar B cells which share phenotypic, morphologic and cytochemical features with subepithelial (SE) B cells. These cells, which represented the 5-10% of the total tonsillar B cells, were found in the Percoll gradient fraction of highest density, together with resting follicular mantle (FM) B cells. The latter B cells, however, expressed surface CD5 and could be removed by an immune rosetting procedure. The remaining small CD5- B cells had a surface phenotype (IgM+, IgD+, CD23-, CD38+/-, CD10-, CD44+) that was different from that of FM (IgM+, IgD+, CD23+, CD39+, CD38-, CD10-, CD44+2) and of germinal center (GC) (CD23-, CD39-, CD38+, CD10+, CD44+/-, IgG+) B cells isolated from the same cell suspensions. Furthermore, the absence of surface activation markers (CD71 and CD69) and of surface IgG allowed us to distinguish small CD5- B cells from activated and memory cells migrating within Percoll fractions of lower density. In situ immunohistochemical studies revealed that B cells with an identical phenotype as that of small CD5- B cells could be detected predominantly in the SE region (lamina propria) of the tonsil, and also within the epithelium lining the cryptae. This area was also comprised of a relatively minor proportion of activated B cells, not found in the small CD5- B cell fraction owing to the separation procedure used. Consistent with the notion that the SE area could be a site of B cell activation was also the presence of activated macrophages and of plasma cells. Thirty to forty percent of small CD5- B cells isolated in suspension were positive for the endogeneous alkaline phosphatase (ALP) activity. In contrast, only a few FM B cells were ALP+, while GC cells were consistently ALP-. In situ studies also demonstrated a prevalent expression of ALP activity by the B cells in the SE area. At the ultrastructural level, small CD5- B cells were clearly different from both FM and GC B cells. They displayed a cytoplasm more extended than that of FM B cells with abundant endosomes and plasma membrane projections, and a speckled pattern of nuclear heterochromatin distribution. When fixed tissue sections were examined, cells with identical ultrastructural features could be demonstrated in the tonsillar lamina propria. Collectively, the above data demonstrate an identity of features between the small CD5- B cells isolated in suspension and SE B cells analyzed in situ. Since tonsillar SE B cells are generally thought to represent the homolog of the extrafollicular B cells (including those of the splenic marginal zone), these studies may provide new opportunities for functional studies on this so far incompletely characterized B cell subset.