This paper investigated transmucosal gas exchange in middle ear (ME) pressure regulation mechanisms. In volunteers with normal ears, positive or negative pressure was applied by Valsalva's or Toynbee's maneuver and ME pressure change without transtubal pressure regulation was monitored for 30-40 minutes. ME pressure tends to return to atmospheric pressure independent of applied pressure indicating that transmucosal gas exchange is involved in ME pressure regulation, together with transtubal pressure regulation. Furthermore, we developed a test to evaluate the transmucosal gas exchange capacity and compared normal ears and ears with otitis media with effusion (OME). ME cavity was insufflated with various gases with different compositions (room air, 100% N2, 100% O2 mixed gas with 5% CO2 and 95% O2). The ME pressure changes due to gas diffusion into or out of the mucosa were measured using a pressure transducer connected to the ME cavity. When room air and 100% N2 were insuffiated into the ME cavity, positive pressure were observed in both normal and OME cases. However, insufflation with 100% O2 or a mixture of 5% CO2 and 95% O2 produced quite different results. In contrast to positive pressure by 100% O2 and no significant pressure change by mixed gas in normal cases, remarkable negative pressure changes were observed in OME ears. This result suggests that O2 absorption is accelerated in cases of OME compared to that in normal ears.From our study, we conclude that the transmucosal gas diffusion in OME cases is quite different from that in the normal ears, and that increased 02 absorption in OME may also promote development of negative ME pressure which is thought to play an important role in the pathogenesis of OME.