The three-dimensional solution structure of apo rabbit lung calcyclin has been refined to high resolution through the use of heteronuclear NMR spectroscopy and 13C, 15N-enriched protein. Upon completing the assignment of virtually all of the 15N, 13C and 1H NMR resonances, the solution structure was determined from a combination of 2814 NOE-derived distance constraints, and 272 torsion angle constraints derived from scalar couplings. A large number of critical inter-subunit NOEs (386) were identified from 13C-select, 13C-filtered NOESY experiments, providing a highly accurate dimer interface. The combination of distance geometry and restrained molecular dynamics calculations yielded structures with excellent agreement with the experimental data and high precision (rmsd from the mean for the backbone atoms in the eight helices: 0.33 A). Calcyclin exhibits a symmetric dimeric fold of two identical 90 amino acid subunits, characteristic of the S100 subfamily of EF-hand Ca(2+)-binding proteins. The structure reveals a readily identified pair of putative sites for binding of Zn2+. In order to accurately determine the structural features that differentiate the various S100 proteins, distance difference matrices and contact maps were calculated for the NMR structural ensembles of apo calcyclin and rat and bovine S100B. These data show that the most significant variations among the structures are in the positioning of helix III and in loops, the regions with least sequence similarity. Inter-helical angles and distance differences for the proteins show that the positioning of helix III of calcyclin is most similar to that of bovine S100B, but that the helix interfaces are more closely packed in calcyclin than in either S100B structure. Surprisingly large differences were found in the positioning of helix III in the two S100B structures, despite there being only four non-identical residues, suggesting that one or both of the S100B structures requires further refinement.