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Anesthesia affects central auditory processing. However, it is unclear to what extent the choice of anesthetic agent affects neural responses to sound stimulation. A mixture of three anesthetics (medetomidine, midazolam and butorphanol; MMB) was recently developed as an alternative to ketamine owing to the latter's addictive potential, yet the effect of this combination of anesthetics on neural responses is not known. Here, we compared the spontaneous activity, tuning properties and temporal responses of primary auditory cortical neurons under these two anesthetic conditions, using electrophysiological and flavoprotein autofluorescence imaging methods. Frequency tuning properties were not significantly different between ketamine and MMB anesthesia. However, neural activity under MMB showed decreases in the spontaneous and tone-evoked firing rates in a layer-dependent manner. Moreover, the temporal response patterns were also different between the anesthetics in a layer-dependent manner, which may reflect differences in the anesthetic mechanisms. These results demonstrated how response properties in the primary auditory cortex are affected by the choice of anesthesia.We compared effects of ketamine and alternative anesthesia in the auditory cortex.Spontaneous and tone-evoked firing rates differed in a laminar-dependent manner.Effects on tuning properties were similar between anesthetics.Anesthetic choice should be carefully considered in auditory processing research.Combining flavoprotein fluorescence imaging with electrophysiology is useful.