The origin and function of thymic B cells is currently unresolved. In the present study we compared V(H) gene repertoire diversification in >3500 cloned VDJs (from 11 animals at three time-points, using three to five animals per time-point) that were expressed with immunoglobulin (Ig)M, IgD, IgG, IgA and IgE in thymus, mesenteric lymph nodes (MLN) and peripheral blood B cells (PBB) of newborn piglets and 5-week-old isolator piglets maintained germfree (GF) or colonized with Escherichia coli. The results showed that the repertoire expressed with IgM, IgD, IgG and IgA in PBB and MLN remained polyclonal, undiversified and unselected in piglets maintained GF for 5 weeks, that age and colonization resulted in significant repertoire diversification of IgG and IgA in the MLN and of IgG in blood, that the thymic B-cell repertoire was polyclonal, unaffected by colonization and showed no clonal selection in any isotype, and that the thymic IgA and IgE repertoires were more diverse at birth than the repertoire of any isotype in MLN or PBB. IgD was seldom recovered from the PBB of newborn piglets or at any time-point in thymus, but was recovered in the MLN of all 11 animals examined. The IgD and IgM repertoires in all tissues remained polyclonal and unselected, although V(H) usage by IgD transcripts did not always parallel that of IgM in the same tissue. Therefore, isotype-switched B cells in the thymic medulla cannot be accounted for by immigration of B cells diversified by colonization of the gut, and thymic B cells undergo switch recombination and repertoire diversification before birth without clonal selection.