Previous data on the genetic origin of insulin Abs in BALB/c mice are derived from immune responses following a single or primary immunization. This primary response, however, may not represent the repertoire established by chronic insulin administration, which is used in the treatment of diabetes mellitus and in protocols to alter beta cell destruction in pre-insulin-dependent (type 1) diabetes mellitus. In this study, the genetic composition and structure of 16 anti-insulin mAbs from the secondary response of a single BALB/c mouse were determined. In contrast to studies on other protein Ags, the V genes in the secondary response to insulin show only a low level of somatic mutation and the majority of the response is represented by restricted sets of V(H) and Vkappa genes. The restriction of V genes is not chiefly due to oligoclonal expansion but reflects recurrent usage of structurally similar V regions of independent origin. Secondary anti-insulin V(H) genes in 12 of 16 mAbs are derived from related J558 subsets, and 8 of 16 mAbs used the Vkappa19.34 gene without mutations in CDRLs. The CDHR3s of half of the mAbs are generated from an uncommon second reading frame of DSP2, suggesting that the amino acid motif STMIT may contact insulin and contribute to selection of these B cells. A total of 14 of 15 mAbs bind autologous rodent insulin with 40 to 100% of the activity on human insulin, and all mAbs bind proinsulin. These data show that a preferred set of V(H), D, and Vkappa gene segments dominate secondary responses to insulin and suggest that specific structural interactions with epitopes that are shared with autologous insulin shape this secondary response.