The auditory steady-state response (ASSR) is a type of auditory evoked potentials, which are electrical activities of the auditory system in the presence of sounds and recorded with electrodes from the scalp. Research has suggested that the ASSR is a useful tool for evaluating the function of the auditory system and is superior to conventional techniques in estimating hearing thresholds in certain special populations. Negative middle ear pressure (MEP) is one of the most common pathologies in humans, particularly among young children. A negative MEP causes retraction of the eardrum which affects the transmission of sounds. However, impact of this condition on the ASSR has never been investigated. The purpose of this study was to make a preliminary observation on ASSR recordings in ears with negative MEP. In 25 human subjects, a positive ear canal pressure (200 daPa) was applied to simulate a negative MEP. The effect of negative MEP is expressed by the change of ASSR amplitude caused by the air pressure. Results show that ASSR amplitudes for all four frequencies were reduced by approximately 12% to 51%, which became larger with increasing frequency from 500 to 4000 Hz.