The Rinne tuning fork test for the detection of conductive hearing loss is the most widely used tuning fork test in clinical medicine. However, the frequency of the tuning fork used to perform the test has not been standardized. In a study of 200 patients with known air-bone gaps, we found that the 256-Hz tuning fork was more sensitive than the 512-, 1024-, and 2048-Hz tuning forks for discriminating conductive from sensorineural hearing deficits. However, the occurrence of false-positive responses limits its usefulness. Therefore, we believe that the 512-Hz tuning fork should become the standard tuning fork used in performance of the Rinne test to screen patients for conductive hearing loss.