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This study was intended to systematically examine the effect of negative middle ear pressure (MEP) on distortion product otoacoustic emissions (DPOAEs) and to validate a compensation procedure to account for negative MEP encountered in DPOAE measurement.In experiment 1, the 2f1 − f2 DPOAE was measured for nine f2 frequencies from 600 to 8000 Hz in 16 adults under three MEP conditions: normal MEP, negative MEP, and compensated MEP. The subjects' voluntarily induced negative MEPs, with magnitudes ranging from −40 to −420 daPa, were measured tympanometrically with the tympanometric peak pressure. Each negative MEP was then compensated for by applying an equivalent amount of negative air pressure into the ear canal. The three MEP conditions were compared in terms of difference in DPOAE level. Experiment 2 was conducted to measure the DPOAE under normal and negative MEP conditions by using a different system with a higher frequency resolution in 19 subjects.Negative MEP generally attenuated DPOAEs more for low frequencies than for high frequencies. For the frequencies of 1000 Hz and below, the mean DPOAE level was reduced by at least 4 to 6 dB for negative MEPs lower than −100 daPa (i.e., less negative). Reduction of the DPOAE level increased with increasing negative MEP (e.g., 10 to 12 dB for −160 daPa and higher, i.e., more negative). For f2 = 2000, 4000, and 6000 Hz, the effect of negative MEP was not significant. For 3000 Hz, DPOAE-level reduction was significant (e.g., 5 dB for MEP = −70 to −95 daPa and up to 12 dB for −290 to −420 daPa). As a result, a peak at 2000 Hz and a notch at 3000 Hz appeared in the DPOAE change versus frequency function. For 8000 Hz, DPOAE levels tended to increase in high negative MEPs, although the changes were not significant. Intersubject variability in the effect of negative MEP on DPOAEs was large. As the negative MEPs were compensated for, the decreased DPOAE levels were significantly corrected. DPOAEs measured with higher resolution in experiment 2 verified the frequency-specific effects of negative MEPs. Results revealed that the peak and notch in the DPOAE change versus frequency function shifted toward higher frequencies when negative MEP was increased, and a second peak emerged at a higher frequency.Negative MEP substantially decreases DPOAE level for low frequencies and some mid-frequencies but tends to increase DPOAE level for high frequencies. Results suggest that any degree of negative MEP should be corrected to obtain an accurate outcome of DPOAE measurement. The MEP compensation procedure is effective in restoring normal DPOAEs in ears with negative MEPs. Examining changes in DPOAE level under negative MEP allows for further study of the transmission of acoustic signals through an altered middle ear system. A minimal change of DPOAE level at 2000 Hz indicates that the primary resonant frequency of the middle ear is lower than 2000 Hz. The variation in DPOAE change in the middle to high frequency range implies multiple resonances of the middle ear system.