Abstract The majority of insulin antibodies derived from immunization are IgG antibodies that cross-react extensively with the autologous hormone. To examine the relationship between VH genes expressed by such self-reactive antibodies and their germline (non-rearranged) counterparts, we used the polymerase chain reaction (PCR) to amplify and isolate the germline progenitors of anti-insulin VH genes derived from BALB/c mice immunized with beef or human insulin. Results indicate that two anti-insulin mAbs (123 and 124) express VH genes which arise from a small subset of the J558 gene family and are highly homologous to the VH gene used by the murine CD5+ B-cell tumor, BCL1. The anti-insulin IgG mAb 127 belongs to the VH-VIII (Vgam 3.2) family and the amplification and isolation of germline VH genes from this small family precisely identified only two somatic mutation events in the CDRH2 of mAb 127. Another anti-insulin mAb, 133, aslo shows two replacement substitutions in the CDRHs when compared to the germline encoded anti-dextran antibody 19.1.2. These findings indicate that the IgG response to this small self-protein uses multiple VH genes which are largely germline encoded with only a low level of somatic mutation in their CDRHs. Additionally, analysis of N-segment additions in CDRH3s indicates anti-insulin B cells may originate from both early (fetal) and adult repertoires. These data are consistent with the concept that the mechanisms of clonal anergy or deletion do not regulate anti-insulin B cells and indicate that there is a large potential VH gene repertoire for insulin.