This study investigates the role of different gases in clearance of gas in the middle ear cavity (ME) by its mucosal blood flow. A rat model was used to measure gas volume changes in the ME cavity at constant pressure without ventilation. We disturbed the normal gas composition of the ME by filling it with O2 or CO2, measured the consequent changes in gas volume over time and compared these results with previously obtained ones for air and N2. The first 5 min of the primary transient phase (phase I) for O2 or CO2 was characterized by a volume loss decrease of −0.49 ± 0.34 μL and −46.28 ± 8.49 μL, respectively, with volume loss increase for air and N2 differing greatly, at +0.17 ± 0.17 and +2.31 ± 0.81, respectively. The CO2 value of −46.28 μL showed that a volume of gas equivalent to that of the ME cleft volume was eliminated within the first 5 min. In the second phase (phase II), all gases showed a linear decrease in volume, which presumably represents a steady-state gas loss rate. However, the gas loss rate of −0.307 ± 0.170 μL min−1 for O2-filled MEs was significantly higher than the mean of −0.124 μL min−1 for all other gases. We used a previously established mathematical model to calculate the effective ME mucosal blood flow rate under steady-state (phase II) conditions. The blood flow results for O2-filled MEs differed greatly from those of the other gases (89.0 ± 49.28 vs. 26.5 μL min−1, on average), which suggest that the model used to calculate blood flow should be modified if used with O2-filled MEs. Further work should involve a comparison of our method with different methods to verify ME blood flow rate.