Recent evidence suggests that G protein-coupled receptors, especially those linked to G(alpha)(i), contribute to the mechanisms of anesthetic action. Regulator of G protein signaling (RGS) proteins bind to activated G(alpha)(i) and inhibit signal transduction. Genomic knock-in mice with an RGS-insensitive G(alpha)(i2) G184S (G(alpha)(i2) GS) allele exhibit enhanced G(alpha)(i2) signaling and provide a novel approach for investigating the role of G(alpha)(i2) signaling and RGS proteins in general anesthesia.We anesthetized homozygous G(alpha)(i2) GS/GS and wild-type (WT) mice with isoflurane and quantified time (in seconds) to loss and resumption of righting response. During recovery from isoflurane anesthesia, breathing was quantified in a plethysmography chamber for both lines of mice.G(alpha)(i2) GS/GS mice required significantly less time for loss of righting and significantly more time for resumption of righting than WT mice. During recovery from isoflurane anesthesia, G(alpha)(i2) GS/GS mice exhibited significantly greater respiratory depression. Poincaré analyses show that GS/GS mice have diminished respiratory variability compared with WT mice.Modulation of G(alpha)(i2) signaling by RGS proteins alters loss and resumption of wakefulness and state-dependent changes in breathing.