In Activated Sludge Model No.3 (ASM3), it is hypothesized that all heterotrophic microorganisms (X(H)) can store substrate. However, in reality, both microorganisms with and without substrate storage capability (X(H/STO) and X(H/S), respectively) could exist. If the ratio of X(H/STO) in activated sludge is influenced by operational and environmental conditions, kSTO (storage rate constant of heterotrophic microorganisms which is defined in ASM3) may not be a universal parameter and can change from case to case. In this study, polyhydroxyalkanoate (PHA) is assumed as the principal storage product, and the ratio of microorganisms with PHA production capability (X(H/PHA)) in various activated sludges was estimated by the dual staining of Nile Blue A (NB) and DAPI. It was shown that the ratio of X(H/PHA) in sludge varied among different municipal and laboratory activated sludges.