The activities of most proteins are relatively insensitive to general anesthetics. A notable exception is firefly luciferase, whose sensitivity to a wide range of anesthetic agents closely parallels that of whole animals. We have now found that this sensitivity can be controlled by ATP. The enzyme is insensitive at low (microM) concentrations of ATP and very sensitive at high (mM) concentrations. The differential sensitivity varies from anesthetic to anesthetic, being greatest (about a 100-fold difference) for molecules with large apolar segments. This suggests that anesthetic sensitivity is modulated by changes in the hydrophobicity of the anesthetic-binding pocket. Parallel changes in the binding of the substrate firefly luciferin, for which anesthetics compete, indicate that anesthetics bind at the same site as the luciferin substrate. These changes in the nature of the binding pocket modify not only the sensitivity to anesthetics but also the position of the "cutoff" in the homologous series of primary alcohol anesthetics; the cutoff position can vary from octanol to pentadecanol, depending upon the concentration of ATP. Our results suggest that particularly sensitive anesthetic target sites in the central nervous system may possess anesthetic-binding pockets whose polarities are regulated by neuromodulatory agents.