Measurements of fluorescence energy transfer have been performed to determine the distance between the lipid-water interface and the ATP-binding site in the (Ca2+ + Mg2+)-ATPase from sarcoplasmic reticulum. The calculated distance between the donor, FITC bound to the protein (nucleotide binding-site marker), and the acceptor, rhodamine-5'-isothiocyanyldipalmitoylphosphatidylethanolamine (RITC-DPPE) incorporated in the membrane, was in the range of 34-42 A. In addition the distance between the high affinity Ca2+-binding sites and the lipid/water interface has been calculated by luminescence energy transfer from Tb3+ bound to the Ca2+ sites to RITC-DPPE included in the membrane, and it was approx. 10 A.