Gordonia amarae is a right-angled branching filament belonging to the mycolic acid-containing Actinobacteria which is commonly found in many foaming activated sludge wastewater treatment plants. Although studies on different substrates as sole carbon sources by pure cultures of G. amarae have been carried out, none have examined substrate uptake by this organism in situ. Uptake of several hydrophilic and hydrophobic substrates by G. amarae was evaluated in situ using a combination of fluorescence in situ hybridization and microautoradiography. G. amarae could assimilate a range of both hydrophilic and hydrophobic substrates. From the data, G. amarae appears to be physiologically active under aerobic, anaerobic and anoxic condition (NO2 and NO3) for some substrates. This might explain why attempts to control foaming caused by G. amarae using anoxic and anaerobic selectors have been unsuccessful. This study emphasizes that bacteria can behave differently in situ to pure cultures and that it is important to evaluate the in situ physiology of these bacteria if we are to better understand their role in the wastewater treatment process.