The structure of a 39 amino acid proteolytic fragment of rabbit skeletal troponin C containing the fourth Ca(2+)-binding site has been determined by an approach involving nuclear magnetic resonance (NMR) spectroscopy combined with hybrid distance geometry-dynamical simulated annealing calculations. Hydrodynamic and NMR evidence establishes unambiguously that the fragment forms a stable dimer in solution in the presence of excess Ca2+. The calculation of the dimeric structure is based on a total of 1056 experimental restraints comprising 422 interproton distances, 35 phi, 28 psi, and 28 chi 1 torsion angle restraints within each subunit, 30 intermonomer distance restraints, and 6 Ca2+ restraints per subunit. A total of 48 final structures were calculated having an rms deviation about the mean atomic backbone coordinate positions of 1.0 A for residues Asp128-Glu156. The solution structure consists of a dimer of helix-loop-helix motifs related by a 2-fold axis of symmetry. The overall architecture of the dimer is very similar to the C-terminal domain in the crystal structure of chicken skeletal troponin C.