We have used time-resolved fluorescence to study proposed conformational transitions in the Ca-ATPase in skeletal sarcoplasmic reticulum (SR). Resonance energy transfer was used to measure distances between the binding sites of 5-[[2-[(iodoacetyl)amino]ethyl]amino]naphthalene-l-sulfonic acid (IAEDANS) and fluorescein 5-isothiocyanate (FITC) as a function of conditions proposed to affect the enzyme's conformation. When 1.0 ± 0.15 IAEDANS is bound per Ca-ATPase, most (76 ± 4%) of the probes have an excited-state lifetime (τ) of 18.6 ± 0.5 ns, and the remainder have a lifetime of 2.5 ± 0.9 ns. When FITC is bound to a specific site on each IAEDANS-labeled enzyme, most of the long-lifetime component is quenched into two short-lifetime components, indicating energy transfer that corresponds to two donor-acceptor distances. About one-third of the quenched population has a lifetime τ = 11.1 ±2.5 ns, corresponding to a transfer efficiency E = 0.40 ± 0.07 and a donor-acceptor distance R1= 52 ± 3 Å. The remaining two-thirds exhibit lifetimes in the range of 1.2-4.2 ns, corresponding to a second distance 31 Å < R2 < 40 Å. Addition of Ca2+ (in the micromolar to millimolar range), or vanadate (to produce a phosphoenzyme analogue), had no effect on the donor-acceptor distances. Addition of decavanadate results in the quenching of IAEDANS fluorescence but has no effect on the energy-transfer distance. Formation of phosphoenzyme from inorganic phosphate at pH 6.2 produced no change when performed in aqueous solution but did produce a small (6.4 ± 3.2 Å) decrease in R1 when performed in 40% DMSO. The lack of large changes in the energy-transfer distance upon the binding of the ligands studied leads us to propose that the B domain, and perhaps the ATPase as a whole, does not undergo large changes in its tertiary structure upon ligand binding.