The fluorescence of TNP-nucleotides bound to sarcoplasmic reticulum ATPase is enhanced upon formation of phosphorylated enzyme intermediate either with ATP in the presence of Ca2+ or, to a greater extent, with Pi in the absence of Ca2+. Binding of the TNP-nucleotides does not occur if the ATPase is labeled at the active site with fluorescein isothiocyanate. Addition of ADP to the TNP-nucleotide X enzyme complex phosphorylated with Pi causes dissociation of TNP-nucleotide and a proportional reduction in fluorescence. These and other kinetic observations indicate that the TNP-nucleotide exchanges with ADP following enzyme phosphorylation with ATP or occupies the ADP portion of the catalytic site following enzyme phosphorylation with Pi. This interaction with the phosphorylated site results in fluorescence enhancement of the TNP-nucleotide. Comparison of the TNP-nucleotide fluorescence features in different solvents with that of the TNP-nucleotide bound to sarcoplasmic reticulum ATPase indicates that, following phosphorylation, the binding domain excludes solvent molecules and confers restricted mobility to the TNP-nucleotide. Solvent exclusion and substrate immobilization accompany, to a greater extent, phosphorylation of the active site with Pi in the absence of Ca2+. TNP-nucleotides bound to the catalytic sites were also found to be acceptors of resonance energy transfer from enzyme tryptophan in the extramembranous domain of the ATPase which also contains the catalytic site.