General anesthetics, with sevoflurane (SF) being the first choice inhalational anesthetic agent, provide reversible, broad depressor effects on the nervous system yet have a narrow margin of safety. As characterization of low-affinity binding interactions of volatile substances is exceptionally challenging with the existing methods, none of the numerous cellular targets proposed as chief protagonists in anesthesia could yet be confirmed. The recognition that most critical functions modulated by volatile anesthetics are under the control of intracellular Ca2+ concentration, which in turn is primarily regulated by calmodulin (CaM), motivated us for characterization of the SF–CaM interaction. Solution NMR (Nuclear Magnetic Resonance) spectroscopy was used to identify SF-binding sites using chemical shift displacement, NOESY and heteronuclear Overhauser enhancement spectroscopy (HOESY) experiments. Binding affinities were measured using ITC (isothermal titration calorimetry). SF binds to both lobes of (Ca2+)4-CaM with low mmol/L affinity whereas no interaction was observed in the absence of Ca2+. SF does not affect the calcium binding of CaM. The structurally closely related SF and isoflurane are shown to bind to the same clefts. The SF-binding clefts overlap with the binding sites of physiologically relevant ion channels and bioactive small molecules, but the binding affinity suggests it could only interfere with very weak CaM targets.