The monoclonal IgM cold agglutinins that bind to the I/i carbohydrate Ags on the surface of RBCs all have Ig H chains encoded by the V4-34 gene segment. This mandatory use indicates that distinctive amino acid sequences may be involved in recognition. Critical amino acids exist in framework region 1 (FR1) of V4-34-encoded Ig, and these generate a specific Id determinant which apparently lies close to the I binding site. However, I binding by Id-expressing Ig can be modulated by sequences in complementarity-determining region (CDR) H 3. Examination of the crystal structure of an anti-I cold agglutinin has revealed a hydrophobic patch in FR1 involving residue W7 on β-strand A and the AVY motif (residues 23–25) on β-strand B. In this study we used mutagenesis to show that each of the strand components of the hydrophobic patch is required for binding the I carbohydrate Ag. In addition, the crystal structure reveals that amino acids in the carboxyl-terminal region of CDR H 3 form a surface region adjacent to the hydrophobic patch. We propose that the I carbohydrate Ag interacts simultaneously with the entire hydrophobic patch in FR1 and with the outside surface of CDR H 3. This interaction could leave most of the conventional binding site available for binding other Ags.