Partial pressures of the gases in the middle ears of 14 guinea pigs were measured continuously on-line with a specially designed mass spectrometer. The average values were carbon dioxide 67.55 mm Hg, oxygen 48.91 mm Hg, and nitrogen 596.54 mm Hg. These values confirm earlier measurements and show that the gas composition of the middle ear differs basically from that of air and resembles that of venous blood. These findings are indicative of bilateral diffusion between the middle ear cavity and the blood. We propose that under physiologic as well as under pathologic (ie, atelectatic) conditions, the gas content of the middle ear is also controlled by diffusion. This mechanism fits well with the fluctuating character of atelectatic ears. Thus, a negative middle ear pressure could be secondary to excessive loss of gases through increased and excessive diffusion, although additional mechanisms are probably also involved. A likely contributing factor is poor pneumatization of the mastoid, with consequent absence of a physiologic pressure regulation mechanism by its pneumatic system.