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Calbindin D 28k , a member of the troponin C superfamily of calcium-binding proteins, contains six putative EF hand domains but binds only four calcium-atoms: one at a binding site of very high affinity and three calcium-atoms at binding sites of lower affinity. The high-affinity site could be located within domain I while domains III, IV, and V bind calcium less tightly. The recombinant protein construct calb I-II (residues 1±93) comprising the first two EF hands affords a unique opportunity to study a pair of EF hands with one site binding calcium tightly and the second site empty. A series of heteronuclear 2D, 3D and 4D high-resolution NMR experiments were applied to calb I-II, and led to the complete assignment of the 1 H, 13 C and 15 N resonances. The secondary structure of the protein was deduced from the size of the 3 J HN-Ha coupling constants, the chemical shift indices of 1 H a , 13 C a , 13 C H and 13 C b nuclei and from an analysis of backbone NOEs observed in 3D and 4D NOESY spectra. Four major a-helices are identified: Ala13±Phe23, Gly33±Ala50, Leu54±Asp63, Val76±Leu90, while residues Ala2±Leu6 form a fifth, flexible helical segment. Two short b-strands (Tyr30±Glu32, Lys72±Gly74) are found preceding helices B and D and are arranged in an anti-parallel interaction. Based on these data a structural model of calb I-II was constructed that shows that the construct adopts a tertiary structure related to other well-described calcium-binding proteins of the EF-hand family. Surprisingly, the protein forms a homodimer in solution, as was shown by its NMR characterization, size-exclusion chromatography and analytical ultra-centrifugation studies.Keywords: calbindin D 28k ; calcium binding protein; EF hand; NMR spectroscopy; secondary structure.Calbindin D 28k is an intracellular calcium binding protein of molecular mass 28 kDa that may be involved in transcellular calcium transport and may modulate effects occurring in response to changes in intracellular calcium concentrations. It was originally purified from chicken intestine [1] and is also expressed in kidney cells. The presence of the protein in numerous additional avian and mammalian tissues suggests an important physiological function [2]. The finding that it is particularly abundant in specific brain regions has led to studies investigating its particular role in calcium homeostasis [3,4].Calbindin D 28k belongs to a family of calcium-binding proteins including many well-known proteins such as calbindin D 9k , troponin C, S100B, calmodulin and calretinin. They are structurally characterized by one or more pairs of a helix-loophelix motif called the EF hand [5] in which five hydrophilic residues and one water molecule bind one calcium atom in a pentagonal bipyramidal coordination via oxygen atoms. The calcium-binding loop is flanked by two a-helices which have a number of residues with hydrophobic side chains that interact with a second EF hand domain forming a hydrophobic core. An approximate twofold symmetry axis relates the pair of EF hand domains....