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_HDJ_H‐μ 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.