We used time‐dependent fluorescence energy transfer to determine the distribution of donor‐to‐acceptor distances in native and denatured troponin I(TnI). The single tryptophan residue (Trp 158) of TnI served as the donor (D), and the acceptor (A) was a labeled cysteine residue (Cys 133). The time‐dependent intensity decays of the donor were measured by the frequency‐domain method from 10 to 320 MHz. The frequency response of the donor emission, in the absence and presence of acceptor, was used to recover the distribution of D to A distances, using an algorithm that accounts for the intrinsic multiexponential decay of the donor. In the native state the D–A distribution is characterized by an average distance of 23 Å and a half‐width of 12 Å. Denaturation results in a modest increase in the average distance to 27 Å, and a dramatic increase in half‐width to 47 Å. We believe the ability to recover distance distributions will have numerous applications in the characterization of biological macromolecules.