Multifrequency tympanometry (MFT), using probe frequencies ranging from 226-2,000 Hz, was performed on normal chinchillas to obtain normative data against which to compare results from animals with middle ear pathology. A series of validating experiments was conducted to determine the effects of anatomical alterations of the middle ear on MFT. These included artificially extending the ear canal, opening the bulla, injecting saline into the middle ear, and disrupting the ossicular chain. The results indicate that MFT characteristics of chinchilla ears are qualitatively similar to those observed in normal humans and patients with middle ear disease, and MFT provides information that is not available from the 226-Hz tympanogram.