Although the Ig H chains of anti-nuclear Abs (ANA) have been described to possess certain shared molecular signatures, it remains unclear whether the L chains of these Abs also possess distinctive molecular features. The present study examines this by generating and analyzing two comprehensive murine Ig L chain databases, one consisting of 264 monoclonal ANAs and the other consisting of 145 non-ANAs, drawn from previously published work. Importantly, clonal replicates were represented only once each, so as to minimize bias. ANAs and non-ANAs did not differ in Vkappa family or Jkappa gene usage, nor in their mutation frequencies. Interestingly, the L chains of ANAs exhibited differential usage of certain complementarity-determining region residues, arising almost entirely from the increased usage of certain Vkappa germline genes, notably, Vkappa ai4 among anti-dsDNA ANAs, Vkappa23-45 among anti-ssDNA ANAs, and Vkappa21-12 among non-ANAs. Finally, prompted by the increased prevalence of a particular Vkappa1 family sequence among ANAs, we proceeded to clone a novel New Zealand Black Vkappa1 germline gene, named bb1.1, which appears to be frequently used to encoded anti-ssDNA Abs. Collectively, these studies underline the potential contribution of particular Vkappa germline genes in promoting or thwarting DNA binding.