Objectives:The primary goal of this study was to evaluate alternative tympanometric parameters for distinguishing normal middle ears from ears with otosclerosis. A secondary goal was to provide guidelines and normative data for interpreting multifrequency tympanometry obtained using the Virtual 310 immittance system.Design:Nine tympanometric measures were examined in 68 normal ears and 14 ears with surgically confirmed otosclerosis. No subjects in either group had a history of head trauma or otoscopic evidence of eardrum abnormalities. Two parameters, static admittance and tympanometric width, were derived from standard low-frequency tympanometry and two parameters, resonant frequency and frequency corresponding to admittance phase angle of 45° (F45°), were derived from multifrequency tympanometry.Results:Differences between normal and otosclerotic ears were statistically significant only for resonant frequency and F45°. Group differences in resonant frequency were larger when estimated using positive tail, rather than negative tail, compensation. Group differences in both resonant frequency and F45° were larger when estimated from sweep frequency (SF), rather than sweep pressure, tympanograms. Test performance analysis and patterns of individual test performance point to two independent signs of otosclerosis in the patient group; 1) an increase in the stiffness of the middle ear, best indexed by F45° derived from SF recordings, and 2) a change in the dynamic response of the tympanic membrane/middle ear system to changes in ear canal pressure, best indexed by tympanometric width. Most patients were correctly identified by only one of these two signs. Thus, optimal test performance was achieved by combining F45° derived from SF recordings and tympanometric width.Conclusions:The findings confirm the advantage of multifrequency tympanometry over standard low-frequency tympanometry in differentiating otosclerotic and normal ears. Recommendations for interpreting resonant frequency and F45° measures obtained using the Virtual Immittance system are also provided. In addition, the relationship among different tympanometric measures suggests a general strategy for combining tympanometric measures to improve the identification of otosclerosis.