The efficiency of denitrification and enhanced biological phosphorus removal in biological nutrient removal activated sludge systems is strongly dependent on the availability of appropriate carbon sources. Due to high costs of commercial compounds (such as methanol, ethanol, acetic acid, etc.) and acclimation periods (usually) required, the effective use of internal substrates is preferred. The aim of this study was to determine the effects of slowly biodegradable compounds (particulate and colloidal), as internal carbon sources, on denitrification, phosphate release/uptake and oxygen utilization for a full-scale process mixed liquor from two large wastewater treatment plants located in northern Poland. Since it is difficult to distinguish the effect of slowly biodegradable substrate in a direct way, a novel procedure was developed and implemented. Four types of one- and two-phase laboratory batch experiments were carried out in two parallel reactors with the settled wastewater without pre-treatment (reactor 1) and pre-treated with coagulation–flocculation (reactor 2). The removal of colloidal and particulate fractions resulted in the reduced process rates (except for phosphate release). The average reductions ranged from 13 % for the oxygen utilization rate during the second phase of a two-phase experiment (anaerobic/aerobic), up to 35 % for the nitrate utilization rate (NUR) during the second phase of a conventional NUR measurement.