The effect of graded variations in middle ear pressure on ossicular vibration was measured in 15 normal human temporal bone specimens. The displacement amplitude of the umbo and stapes head was measured at 16 frequencies between 0.2 kHz and 3.5 kHz at a constant sound pressure of 134 dB SPL at the tympanic membrane (TM) using a non-contacting video measuring system. Both negative and positive pressures decreased umbo and stapes vibration at low frequencies and slightly increased the vibration at higher frequencies. The effects were greater for negative pressure than for positive pressure. The change in stapes vibration was less than that of the umbo at low frequencies, but increased at higher frequencies. In some temporal bones, a small positive pressure produced improvement in stapes vibration at all frequencies. These effects were thought to be primarily due to an increased stiffness of the TM and a damping of ossicular vibration, due to stretching of the ossicular suspensory ligaments and the annular ligament of the footplate.