Computational methods designed to predict and visualize ligand protein binding interactions were used to characterize volatile anesthetic (VA) binding sites and unoccupied pockets within the known structures of VAs bound to serum albumin, luciferase, and apoferritin. We found that both the number of protein atoms and methyl hydrogen, which are within ∼8 Å of a potential ligand binding site, are significantly greater in protein pockets where VAs bind. This computational approach was applied to structures of calmodulin (CaM), which have not been determined in complex with a VA. It predicted that VAs bind to [Ca2+]4-CaM, but not to apo-CaM, which we confirmed with isothermal titration calorimetry. The VA binding sites predicted for the structures of [Ca2+]4-CaM are located in hydrophobic pockets that form when the Ca2+ binding sites in CaM are saturated. The binding of VAs to these hydrophobic pockets is supported by evidence that halothane predominantly makes contact with aliphatic resonances in [Ca 2+]4-CaM (nuclear Overhauser effect) and increases the Ca2+ affinity of CaM (fluorescence spectroscopy). Our computational analysis and experiments indicate that binding of VA to proteins is consistent with the hydrophobic effect and the Meyer-Overton rule. © 2006 by the Biophysical Society.