AbstractParvalbumins are a class of calcium‐binding proteins characterized by the presence of several helix‐loop‐helix (EF‐hand) motifs. It is suspected that these proteins evolved via intragene duplication from a single EF‐hand. Silver hake parvalbumin (SHPV) consists of three EF‐type helix‐loop‐helix regions, two of which have the ability to bind calcium. The three helix‐loop‐helix motifs are designated AB, CD, and EF, respectively. In this study, native silver hake parvalbumin isoform B (SHPV‐B) has been sequenced by mass spectrometry. The sequence indicates that this parvalbumin is a β‐lineage parvalbumin. SHPV‐B was cleaved into two major fragments, consisting of the ABCD and EF regions of the native protein. The 33‐amino acid EF fragment (residues 76‐108), containing one of the calcium ion binding sites in native SHPV‐B, has been isolated and studied for its structural characteristics, ability to bind divalent and trivalent cations, and for its propensity to undergo metal ion‐induced self‐association. The presence of Ca2+ does not induce significant secondary structure in the EF fragment. However, NMR and CD results indicate significant secondary structure promotion in the EF fragment in the presence of the higher charge‐density trivalent cations. Sedimentation equilibrium analysis results show that the EF fragment exists in a monomer‐dimer equilibrium when complexed with La3+.