Anti-polysaccharide Ab responses in mice are often oligoclonal, and the mechanisms involved in Ag-specific clone production and selection remain poorly understood. We evaluated the relative contribution of DH germline content versus N nucleotide addition in a classic oligoclonal, T-independent Ab response (α 1→3 dextran [DEX]) by challenging adult TdT-sufficient (TdT+/+) and TdT-deficient (TdT-/-) gene-targeted mice, limited to the use of a single DH gene segment (D-limited mice), with Enterobacter cloacae. D-limited mice achieved anti-DEX-specific levels of Abs that were broadly comparable to those of wild-type (WT) BALB/c mice. Sequence analysis of the third CDR of the H chain intervals obtained by PCR amplification of VH domain DNA from DEX-specific plasmablasts revealed the near universal presence of an aspartic acid residue (D99) at the V-D junction, irrespective of the composition of the DH locus. Although WT mice were able to use germline DH (DQ52, DSP, or DST) gene segment sequence, TdT activity, or both to produce D99, all three D-limited mouse strains relied exclusively on N addition. Additionally, in the absence of TdT, D-limited mice failed to produce a DEX response. Coupled with previous studies demonstrating a reduced response to DEX in TdT-/- mice with a WT DH locus, we concluded that in the case of the anti-DEX repertoire, which uses a short third CDR of the H chain, the anti-DEX response relies more intensely on sequences created by postnatal N nucleotide addition than on the germline sequence of the DH. Copyright © 2011 by The American Association of Immunologists, Inc.