Antifreeze proteins lower the freezing point of their solution by binding to ice and inhibiting its growth. One of several structurally different antifreeze proteins in fishes (type II) is homologous to the carbohydrate-recognition domain of Ca²⁺-dependent lectins and adopts the same three-dimensional fold. Type II antifreeze proteins from herring and smelt require Ca²⁺ for binding to ice, whereas this same antifreeze protein in sea raven binds to ice in the absence of Ca²⁺ and has only two of the five Ca²⁺-liganding amino acids that are present in the lectin. To locate the ice-binding site, site-directed mutants of the 15 kDa, globular, disulfide-bonded sea raven antifreeze protein were produced by secretion from Pichia pastoris. Pairs of amino acid replacements, insertions, and a peptide loop swap were made in the region equivalent to the sugar-binding site of the lectin that encompasses loops 3 and 4 and β-sheets 7 and 8. Even the most extensive mutation caused only a 25% decrease in antifreeze activity and demonstrated that the residues corresponding to the Ca²⁺-binding site are only peripherally involved in ice binding. When adjacent surface residues were mutated, the replacement of one residue, Ser120 by His, caused a 35% decrease in activity by itself and an 80% loss in conjunction with the peptide loop swap mutation. This pivotal sea raven antifreeze protein amino acid does not coincide with the herring ice-binding epicenter, but is located within the region corresponding to the proposed CaCO₃-binding surface of a third homologue, the pancreatic stone protein. Intron and exon structure of the sea raven AFP gene also suggests that it might be more closely related to the stone protein gene than to the lectin gene. These results support the notion that this family of proteins has evolved more than one binding surface from the same protein scaffold.