HYPOTHESIS:: Middle ear pressure (MEP) is actively regulated by both the Eustachian tube and the mastoid air cell system. BACKGROUND:: MEP is a highly significant factor involved in many clinical conditions related to otitis media. Basic knowledge on its overall regulation remains insufficient, but the Eustachian tube and mastoid gas exchange are important factors. The main focus has been aimed at the tube; however, evidence points to the mastoid as equally important. More detailed methods are demanded to study their complementary functions. METHODS:: A catheter was inserted into the mastoid of 12 human volunteers (patients for parotidectomy). This enabled monitoring MEP directly, and experiments were performed with volume displacements of +/-50, 100, and 200 mul resulting in a range of overpressure and underpressure. The following counter-regulation was recorded over 10 minutes. RESULTS:: In some cases, MEP counter-regulation presented as Eustachian tube openings with steep and fast pressure changes toward 0 Pa, whereas in others, gradual and slow pressure changes presented related to the mastoid; these changes sometimes crossed 0 Pa into opposite pressures. In many cases, combinations of these distinct mechanisms were found. CONCLUSION:: The human mastoid as well as the Eustachian tube was capable of active counter-regulation of the MEP in short-term experimental pressure changes in healthy ears. Thus, these 2 systems seemed to function in a complementary way, where the mastoid was related to continuous regulation of smaller pressures, whereas the tube was related to intermittent regulation of higher pressures.