In this communication it is reported that the anisotropy of the fluorescence of fluorescein phosphatidylethanolthiocarbamide (FPE) is higher in reconstituted membranes of sarcoplasmic reticulum Ca 2 + -ATPase than in liposomes of phosphatidylcholine or of phosphatidylcholine-phosphatidylserine, and is dependent on the lipid to protein ratio. The dependence of the fluorescence anisotropy of FPE on the molar fraction of annular lipids does not fit the theoretical prediction for random distribution of FPE between annular and bulk lipids. The experimental results are fitted, however, by introducing the effect of preferential binding of FPE over unlabelled lipids to annular sites. The theoretical simulations allow us to conclude that FPE binds to the annular Ca 2 + -ATPase sites with 3- to 7-fold higher affinity than phosphatidylcholine or phosphatidylserine. This study shows the utility of the fluorescence anisotropy of lipids labelled at the polar head group to monitor lipid binding to annular sites of sarcoplasmic reticulum Ca 2 + -ATPase. The methods developed in this paper can also be applied to other reconstituted membranes and to decide whether or not a given fluorescent lipid derivative should be used for measurements of interfacial physical-chemical properties of the lipid bilayer of native biological membranes.