Immune responses by mice to porcine and bovine insulins are controlled by dominant genes linked to the H-2 gene complex. Mice bearing the H-2[d] haplotype respond to porcine insulin, whereas mice bearing other H-2 haplotypes do not; mice bearing the H-2[b,d,v] haplotypes respond to bovine insulin, whereas other strains do not. Analysis of serum antibody by isoelectric focusing demonstrates that antibodies specific for both porcine and bovine insulin are of restricted heterogeneity, suggesting that heterologous insulins stimulate a limited number of B cell clones. Banding patterns (spectrotypes) of sera from individuals of a given responder strain are remarkably uniform and these spectrotypic profiles are strain associated. In addition, phenotypic expression of spectrotypes is codominant in F1 hybrid mice and is controlled principally by allotype-linked genes. Analysis of cross-reactivity patterns demonstrates that all anti-porcine insulin antibodies bind bovine insulin and the majority of anti-bovine insulin antibodies bind porcine insulin. The minority of anti-bovine insulin antibodies that fails to bind porcine insulin may be specific for the A chain loop region of bovine insulin. These observations suggest that murine anti-insulin antibodies will provide a valuable model system for the identification of variable region genes that encode binding sites specific for protein molecules.