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Many studies of the auditory system are performed on animals under general anesthesia. A concern for researchers is that these agents may significantly alter the underlying neurophysiologic mechanisms being studied. The effects may very across species, and even among individuals within a species. An investigation was undertaken to study whether DPOAE measures differ using three different anesthetic regimens: acetylpromazine–ketamine, xylazine–ketamine, and sodium pentobarbital. The same rat was anesthetized in three consecutive weeks using a different anesthetic regimen each week. DPOAE magnitude and phase temporal responses were recorded from which several measures were taken: DPOAE levels at the onset of the primaries, changes in DPOAE level as a function of time during presentation of the primaries (ΔLI) and changes in DPOAE level (ΔLC) and phase (ΔPC) during presentation of a broad-band noise presented contralateral to the probe. Each week the same measurements were repeated with the rat anesthetized using a different regimen and at the end of the third week, the middle ear muscles were sectioned and the measurements repeated once again. Results showed that the anesthetic regimens did not differentially alter the DPOAE onset levels. When sodium pentobarbital was used as the anesthetic regimen, ΔLC and ΔPC were significantly smaller relative to those measured when the rats were anesthetized with acetylpromazine–ketamine and xylazine–ketamine. Based on the assumption that large, positive (ΔPC) values are related to middle ear muscle activation, the middle ear muscle reflex remained at least partially active in some rats under sodium pentobarbital anesthesia. The ΔLI measures were significantly smaller when the animals were anesthetized with xylazine–ketamine and sodium pentobarbital than when they were anesthetized with acetylpromazine–ketamine. Recordings taken after sectioning the middle ear muscles suggested that the middle ear muscle reflex substantially contributes to ΔLC and ΔPC measures under the anesthetic regimens xylazine–ketamine and acetylpromazine–ketamine. Data indicated that anesthetic agents variably alter neurophysiologic mechanisms involved with the complex control of the auditory signal even among individuals in the same species. Extreme care should be taken when comparing ΔLI, ΔLC and ΔPC across studies when different anesthetic regimens are used within and across species.