Activated sludge from the municipal waste water treatment plant in Hamburg was seeded with mineral nitrite medium and incubated at 10°C, 17°C and 28°C. Dominant lithoautotrophic nitrite-oxidizing bacteria have been identified by electron microscopy, denaturing and temperature gradient gel electrophoresis and PCR with genus-specific primer pairs. The results have revealed the existence of three different genera of nitrite-oxidizing bacteria, namely Nitrospira, Nitrobacter and a novel cold-adapted nitrite oxidizer. As shown by electron microscopy members of the novel genus coexisted in activated sludge together with Nitrospira. A temperature-dependent shift in the population structure was demonstrated by cultivation-based approaches. The novel nitrite oxidizer was enriched at temperatures of 10°C and 17°C. Representatives of Nitrospira were able to grow in a broad temperature range between 10°C and 28°C and members of Nitrobacter were enriched during incubations at 17°C and 28°C. By subsequent 16S rDNA sequencing, the cold-adapted nitrite oxidizer was shown to be closely related to the betaproteobacterium ‘Candidatus Nitrotoga arctica’. These findings demonstrated that the population structure of nitrite-oxidizing bacteria in activated sludge is more complex than previously thought and responds strongly to long-term temperature changes.