We have successfully substituted Pu-240(3+) for Ca2+ in the calcium-binding protein calmodulin and used neutron resonance scattering from the bound Pu-240 to demonstrate that the Pu binds specifically to the Ca2+ sites and also to measure the distance between the ion binding sites within individual domains of the protein. Pu-240 has a strong nuclear resonance at 0.278 Angstrom, and at this wavelength the coherent scattering from Pu-240 is > 1000 times that of any other nucleus present in a protein. The ionic radius of Pu3+ is very similar to that of Ca2+, and hence eve chose this species to substitute for Ca2+ in the protein. We identified solution conditions that stabilize Pu3+ in solution at near neutral pH for 6-7 h in order to form the Pu/calmodulin complex under conditions favorable for both complex formation and maintaining the structural integrity of the protein. We collected small-angle neutron scattering data from solutions of 4(Pu-240(3+)). calmodulin, which contain periodic terms that are directly related to the distances between the Ca2+-binding sites. The shorter Pu-Pu distance, i.e., the average distance between the two sites within each globular domain of calmodulin, is found to be 11.8 +/- 0.4 Angstrom, in excellent agreement with the value of 11.7 Angstrom from crystallographic determinations. This is the first use of neutron resonance scattering as a structural probe in a protein.