The V H 4 genes expressed by both resting and in vivo-activated subepithelial (SE) B cells from human tonsils were studied. Resting SE B cells were subdivided according to the presence (IgD low) or absence (IgM-only) of surface IgD. CD27 was abundant on activated SE B cells and low on resting IgM-only B cells. Resting IgD low SE B cells could be subdivided into CD27 low and CD27 high cell fractions. Resting IgD low SE B cells displayed V H 4 genes with a substantial number of mutations (13/29 of the molecular clones were mutated), whereas 25/26 of the clones from resting IgM-only SE B cells were unmutated. Moreover, mutated V H 4 genes were detected mainly within the CD27 high cell fraction of the IgD low SE B cells. Several identical unmutated V H 4DJ H sequences (11/32) were found in different molecular clones from resting IgM-only SE B cells, suggesting local cellular expansion. Both unmutated (14/25) and mutated (11/25) sequences were found in μ transcripts of activated SE B cells. Extensive mutation was observed in the γ transcripts of activated SE B cells. Therefore, SE B cells are heterogeneous, being comprised of B cells with mutated Ig V H 4 genes, that are Ag-experienced B cells, and a subset of B cells with unmutated V H 4 genes that are either virgin cells or cells driven by Ags that did not induce or select for V gene mutations.