The middle ear muscle (MEM) reflexes function to protect the inner ear from intense acoustic stimuli and to reduce acoustic masking. Sound presented to the same side or to the opposite side activates the MEM reflex on both sides. The ascending limbs of these pathways must be the auditory nerve fibers originating in the cochlea and terminating in the cochlear nucleus, the first relay station for all ascending auditory information. The descending limbs project from the motoneurons in the brainstem to the MEMs on both sides, causing their contraction. Although the ascending and descending pathways are well described, the cochlear nucleus interneurons that mediate these reflex pathways have not been identified. In order to localize the MEM reflex interneurons, we developed a physiologically based reflex assay in the rat that can be used to determine the integrity of the reflex pathways after experimental manipulations. This assay monitored the change in tone levels and distortion product otoacoustic emissions within the ear canal in one ear during the presentation of a reflex-eliciting sound stimulus in the contralateral ear. Preliminary findings using surgical transection and focal lesioning of the auditory brainstem to interrupt the MEM reflexes suggest that MEM reflex interneurons are located in the ventral cochlear nucleus. Anat Rec Part A, 2006. © 2006 Wiley-Liss, Inc.