Systemic lupus erythematosus (SLE) and the MRL-lpr/lpr murine model for SLE are characterized by the presence of serum anti–double-stranded (ds)DNA antibodies (Abs), whereas nonautoimmune individuals have negligible levels of these Abs. To increase the frequency of anti-DNA B cells and identify the mechanisms involved in their regulation in nonautoimmune mice, we have used Ig transgenes (tgs). In the present study, we used the VH3H9 heavy (H) chain tg which expresses an H chain that was repeatedly isolated from anti-dsDNA Abs from MRL-lpr/lpr mice. Because the VH3H9 H chain can pair with endogenous L chains to generate anti–single-stranded DNA, anti-dsDNA, and non-DNA B cells, this allowed us to study the regulation of anti-dsDNA B cells in the context of a diverse B cell repertoire. We have identified anti-dsDNA B cells that are located at the T–B interface in the splenic follicle where they have an increased in vivo turnover rate. These anti-dsDNA B cells exhibit a unique surface phenotype suggesting developmental arrest due to antigen exposure.