The moving bed biofilm reactor (MBBR) wastewater treatment process is usually designed based on the assumption that all activity in the process occurs in the biofilm on the MBBR carriers, although there is always some active biomass in the bulk liquid due to biofilm sloughing and, sometimes, free-growing bacteria. In this study the removal of organic matter is evaluated in laboratory-scale MBBR reactors under varying load, hydraulic retention time (HRT), oxygen concentration and volumetric filling degree of carriers in order to determine the heterotrophic activity in the different fractions of the MBBR biomass. The results showed that the heterotrophic conversions in an MBBR can show the same type of diffusion limited dependency on oxygen as nitrification, even for easily degradable substrates such as acetate. The contribution to the removal from the suspended biomass is shown to vary depending on HRT, as the amount of suspended solids changes. The developed method in this report is a useful tool for determining heterotrophic activity in the separate fractions of biomass in MBBRs.