The separation and accurate quantification of active biomass components in activated sludge is of paramount importance in models, used for the management and design of wastewater (WW) treatment plants. Accurate estimates of microbial population concentrations and the direct, in situ determination of kinetic parameters could improve the calibration and. validation of existing models of biological nutrient removal activated sludge systems. The aim of this study was to obtain correlations between heterotrophic active biomass (Z(BH)) concentrations predicted by mathematical models and quantitative information obtained by Fluorescent in situ hybridizations (FISH). Respirometric batch test were applied to mixed liquors drawn from a well-defined parent anoxic/aerobic activated sludge system to quantify the ZBH concentrations. Similarly fluorescent labeled, 16S rRNA-targeted oligonucleotide probes specific for ammonia and nitrite oxidizers were used in combination with DAPI staining to validate the ZBH active biomass component in activate sludge respirometric batch tests. For the direct enumeration and simultaneous in situ analysis of the distribution of nitrifying bacteria, in situ hybridization with oligonucleotide probes were used. Probes (NSO 1225, NSR 1156, and NIT3) were used to target the nitrifiers and the universal probe (EUB MIX) was used to target all Eubacteria. Deducting the lithoautotrophic population from the total bacteria population revealed the ZBH population. A conversion factor of 8.19 x 10(-11) mg VSS/cell was applied to express the ZBH in terms of COD concentration. ZBH values obtained by molecular probing correlated closely with values obtained from the modified batch test. However, the trend of consistently poor correspondence of measured and theoretical concentrations were evident. Therefore, the focus of this study was to investigate alternative technology, such as FISH to validate or replace kinetic parameters which are invariably incorporated into models.