Marginal zone B (MZ-B) cells of the spleen contribute significantly to the immunity against invasive infections with polysaccharide-encapsulated bacteria. Recent evidence indicates that recruitment and selection of MZ-B cells occurs on the basis of positive selection constraints that likely operate via B cell receptor (BCR) signaling. Previous studies have shown that MZ-B cells carry relatively shorter immunoglobulin (Ig) heavy (H) chain complementarity-determining region 3 (H-CDR3) sequences and express BCR which are thought to be polyreactive. In this scenario, MZ-B cell selection proceeds via engagement of the BCR with exogenous (i.e. microbial gut flora-derived) and/or endogenous (self) antigens. Here, we studied the influence of exogenous antigens on the selection process of MZ-B cells using non-genetically manipulated adult germ-free and conventionally reared infant rats. This study was carried out by H-CDR3 spectratype analysis of V(H)(PC7183)-encoded Ig V(H)DJ(H)-mu transcripts expressed by purified splenic MZ-B cells and other B cell subsets. We show that MZ-B cells in both adult germ-free and conventionally reared infant (14-day-old) rats are H-CDR3-selected cells, providing strong evidence that recruitment and selection of MZ-B cells is driven by self antigens.