Both metal cations and organic cations are known to regenerate the purple color and function of bR from its blue deionized nonfunctional form. To understand the role of cations in the function of bR, the difference between the binding properties of organic and metal cations needs to be determined. In this work, the binding constants and the number of RU(bpy)(3)(2+) ions in each site in regenerated bR are determined from Scatchard plots by using fluorescence measurements in the 3.6-4.3 pH range. Three strongly binding sites are identified : the first two sites involve two Ru(bpy)(3)(2+) with the same high-affinity binding constant, and the third one has a medium affinity binding constant. An upper limit on the Ru(bpy)(3)(2+)-retinal distance is estimated from Forster energy-transfer studies. The photocycle of fully regenerated bR is initiated by energy transfer from the excited state of bound RU(bpy)(3)(2+) to the retinal. By monitoring the amount of M(412) intermediate formed by this process relative to that formed in native bR, an energy-transfer efficiency E greater than or equal to 97% is determined. From this low-limit value of E, the average distance between the retinal chromophore and the Ru(bpy)(3)(2+) energy donor(s) in different binding sites is estimated to be less than or equal to 16 Angstrom.