Till now the role of storage in activated sludge processes under transient conditions has been deeply investigated under anaerobic (EBPR processes) or aerobic (bulking control) environments. Little attention has been given to the role of storage in processes including anoxic environments. Hence, the aim of the present work was to investigate the anoxic storage along with other substrate removal mechanisms under transient conditions. Several mixed culture were ad hoc selected under anoxic environment and periodic feeding (acetate as carbon source) at different organic load rate (OLR) and feed length; then their transient response to substrate spike was investigated by batch tests under both anoxic and aerobic conditions. The relative role of different mechanisms in the substrate removal was established on the basis of COD balance assuming that the acetate COD removed from the liquid phase could be oxidised for energy needs or recovered into solids as poly-3-hydroxybutyric acid (PHB) (storage), other internal precursors or intermediates (accumulation) and active biomass (growth, as estimated by ammonium uptake). In all tested conditions, growth response was very little while PHB storage was prevailing. In some operating conditions, indirect evidence of accumulation (in forms still to be identified) was also found. The transient response was not affected by the presence of free amino acids, at least for the unacclimated mixed culture under observation. Transient response under aerobic condition was quite similar to the anoxic one.