Upon excitation by 280 nm, the intensity decay of the 2 tyrosine residues (residues 10 and 109) of rabbit skeletal muscle troponin C is resolved into three components. The anisotropy decay in the absence of divalent cation is biphasic with a short correlation time of 0.67 ns and a long correlation time of 9.23 ns. The limiting anisotropy is 0.225, considerably lower than the value expected for immobilized tyrosine. Upon excitation by 290 nm, the anisotropy decay is also biphasic, and the limiting anisotropy increases to 0.274. The recovery of anisotropy by excitation at a wave-length near the red edge of the tyrosine absorption spectrum is evidence of fluorescence resonance energy transfer between the two tyrosines. For energy transfer to occur, the average separation between the 2 tyrosines is unlikely much larger than the Förster distance Ro, congruent to 10 A, and this close proximity of the residues would require a highly distorted dumbbell shape of troponin C in solution. These results are consistent with a flexible central helix, which either has a segmental flexibility with large amplitude or results in a spectrum of conformations including those in which the two globular domains are in a very close proximity.