Background. Naturally occurring antibodies (Nabs) that bind to terminal galactose α1,3-galactose carbohydrate structures (Gal) are present in humans and Old World monkeys but are negatively regulated in other mammalian species because they express Gal epitopes on their cell surfaces. A Gal knockout mouse (Gal-/-) model, generated by homologous disruption of α1,3-galactosyltransferase gene, is capable of producing natural anti-Gal Abs. Methods. To study the genetic control of the anti-Gal response, we have generated anti-Gal hybridomas from Gal-/- mice and analyzed VH genes of anti-Gal Abs from naive animals and from mice stimulated by rat heterotopic heart transplantation. Results. Six immunoglobulin (Ig)M anti-Gal hybridomas derived from naïve Ga-/- mice exhibited anti-Gal binding activity with some cross-reactivity to related carbohydrate structures. These naïve anti-Gal Abs used five different VH genes in a germline configuration. Anti-Gal IgM hybridomas isolated after a rat heterotopic heart xenograft (4 days) utilized germline VH gene segments from the VH7183 family and exhibited less cross-reactivity. In contrast to mice 4 days after xenograft, we have predominantly isolated IgG antiGal hybridomas from mice 21 days after rat heterotopic heart xenografts, indicating an isotype switch. Nine of the IgG anti-Gal hybridomas secreted IgG3 subclass and one produced IgG1. Sequence analysis of the VH gene usage from the induced anti-Gal IgG antibodies demonstrated a restricted gene utilization (VHJ606-V14A). Conclusion. Our results demonstrate that the antiGal response in naïve Gal-/- mice is encoded by multiple germline progenitors. In response to a xenograft, the induced anti-Gal Abs exhibited a restricted gene usage and somatic mutations, indicating a positive selection.