In the bone marrow, the passage of developing B cells through critical checkpoints of differentiation is associated with a reduction of specific categories of CDR3 of the Ig heavy chain (CDR-H3), particularly those with excessive hydrophobic or charged amino acids and those with a length of eight or fewer residues. To gain insight into the role of CDR-H3 content in the development of B cells in the spleen, we compared the sequences of V(H)7183DJCmu transcripts from sorted transitional T1, marginal zone, and follicular B cell subsets to those expressed by immature IgM(+)IgD(-) and mature IgM(lo)IgD(hi) B cells in the bone marrow. Although differences in V(H) utilization were noted, the T1 CDR-H3 repertoire showed extensive similarity to that of immature bone marrow B cells, and the follicular CDR-H3 repertoire most resembled that of mature bone marrow B cells. Unlike the splenic follicular and bone marrow mature B cell CDR-H3 repertoires, the marginal zone B cell CDR-H3 repertoire retained both short and highly charged amino acid motifs, including those with two arginines. Our findings suggest that antigen binding sites containing specific categories of CDR-H3 sequence content may inhibit, permit, or even facilitate passage of the host B cell through critical checkpoints in peripheral as well as central development.