Analyses based on ribosomal RNA (rRNA)-targeted hybridization performed in our laboratory identified two types of bacterial populations: a population with a high RNA level per biomass and a population with a low level of RNA per biomass. To extend these descriptions, the diurnal dynamics of the RNA pool were monitored by rRNA-targeted oligonucleotide probe membrane hybridization. Under the typical diurnal variation in COD loading rate experienced by municipal wastewater treatment plants, the RNA level of the bacterial population with a high level of RNA per biomass varied with changes in the COD loading rate. Under the same conditions, the RNA level of the population with low RNA level per biomass remained constant. A structured biomass model was developed to describe these data. Substrate COD was divided into a readily biodegradable and a slowly biodegradable COD fraction. It was assumed that two specialized populations coexist in municipal activated sludge treatment systems. One population consumes readily degradable COD and the other consumes slowly degradable COD. According to model simulations, the population consuming readily degradable COD has a high level of RNA per biomass under variable substrate concentrations. Comparatively, the population consuming slowly degradable COD has a low level of RNA level per biomass. Furthermore, model simulations reproduced the two diurnal RNA profiles observed in a full-scale municipal activated sludge system. Therefore, we suggest that two populations can be distinguished in municipal activated sludge systems: a population consuming readily degradable substrate and a population consuming slowly degradable substrate.