Ammonia-oxidizing microorganisms (AOM) generate their energy by the oxidation of ammonia (NH3) to nitrite (NO2−). This process can be carried out by ammonia-oxidizing bacteria (AOB) as well as by the recently discovered ammonia-oxidizing archaea (AOA). In the past, AOB were enriched in batch cultures, often in the presence of rather high concentrations of NH4+. Here, we describe methods to enrich, isolate, and investigate the basic physiology of AOB and AOA with emphasis on those that are adapted to low NH4+ concentrations. The methods described include enrichment of AOA and AOB in batch cultures and of AOB in continuous cultures, the isolation of AOA by serial dilution and AOB by pour plates or dilution to extinction, and techniques to determine growth and activity of the AOA and AOB. Finally, we incorporated a section with Appendix about the identification of AOA and AOB as well as the measurement of the different inorganic nitrogen species.