Multiple impeller reactors are widely used due to their advanced gas utilization and an increased volumetric mass transfer coefficient. However, with the application of Rushton impellers, gas dispersion efficiency varies between the bottom and the upper impeller levels. The present study analyzes the individual flow regime, power input and gas holdup in each compartment of a reactor equipped with four Rushton impellers. The results indicate that the pre-dispersion of the air introduced by the bottom impeller plays a key role in a better gas retention efficiency of the upper impellers. In contrast, a flooded bottom impeller adversely affects the gas dispersion of all impellers. A novel analysis of the bubble flow in the dispersed state via a two-phase CFD model reveals that a more homogenous distribution of air bubbles in the upper compartments leads to high compartment gas holdup values, but fewer bubbles in the vicinity of the impellers. The measured and simulated data of this study indicate that the upper impellers' efficiency mostly depends on the flow regime of and the pre-dispersion by the bottom impeller rather than on the upper impellers' flow regimes. These results contribute to the understanding of essential mixing processes and scaling of aerated bioreactors.