This paper characterizes the low-affinity ligand binding interactions of a fluorinated volatile anesthetic, isoflurane (CHF2OCHClCF3), with bovine serum albumin (BSA) using 19F-NMR transverse relaxation (T2). 19F-NMR spectra of isoflurane in aqueous BSA reveal a single isoflurane trifluoromethyl resonance, indicative of rapid exchange of isoflurane between protein-bound and aqueous (free) environments. The exchange is slow enough, however, that the chemical shift difference between bound and free isoflurane (δω) = 0.545 ppm) contributes to the observed isoflurane T2. The contribution of δω to T2 can be minimized by shortening the interval between 180° refocusing pulses in the Carr-Purcell-Meiboom-Gill pulse sequence used to monitor T2. Analysis of the dependence of T2 on interpulse interval additionally allows determination of the T2 (6.2 ms) and the average lifetime (τb =187 µs) of bound isoflurane molecules. By use of a short interpulse interval (<100 ms), 72 measurements can readily be used to analyze equilibrium binding of isoflurane to BSA. This analysis revealed a discrete saturable binding component with KD = 1.4 mM that was eliminated either by coincubation with oleic acid (6 mol/mol of BSA) or by conversion of BSA to its “expanded”form by titration to pH 2.5. The binding was independently characterized using a gas chromatographic partition analysis (KD =1.4 mM, Bmax = 3-4 sites). In summary, this paper describes a method whereby T2 measurements can be used to characterize equilibrium binding of low-affinity ligands to proteins without the confounding contributions of chemical shift. Additionally, this paper directly demonstrates that distinct volatile anesthetic binding sites exist on certain proteins, in certain conformations, and provides a sensitive method to study them.