Both bacteria and archaea potentially contribute to ammonia oxidation, but their roles in freshwater sediments are still poorly understood. Seasonal differences in the relative activities of these groups might exist, since cultivated archaeal ammonia oxidizers have higher temperature optima than their bacterial counterparts. In this study, sediment collected from eutrophic freshwater Lake Taihu (China) was incubated at different temperatures (4°C, 15°C, 25°C, and 37°C) for up to 8 weeks. We examined the active bacterial and archaeal ammonia oxidizers in these sediment microcosms by using combined stable isotope probing (SIP) and molecular community analysis. The results showed that accumulation of nitrate in microcosms correlated negatively with temperature, although ammonium depletion was the same, which might have been related to enhanced activity of other nitrogen transformation processes. Incubation at different temperatures significantly changed the microbial community composition, as revealed by 454 pyrosequencing targeting bacterial 16S rRNA genes. After 8 weeks of incubation, [13 C]bicarbonate labeling of bacterial amoA genes, which encode the ammonia monooxygenase subunit A, and an observed increase in copy numbers indicated the activity of ammonia-oxidizing bacteria in all microcosms. Nitrosomonas sp. strain Is79A3 and Nitrosomonas communis lineages dominated the heavy fraction of CsCl gradients at low and high temperatures, respectively, indicating a niche differentiation of active bacterial ammonia oxidizers along the temperature gradient. The 13 C labeling of ammonia-oxidizing archaea in microcosms incubated at 4 to 25°C was minor. In contrast, significant 13 C labeling of Nitrososphaera-like archaea and changes in the abundance and composition of archaeal amoA genes were observed at 37°C, implicating autotrophic growth of ammoniaoxidizing archaea under warmer conditions. I nland waters supply essential ecosystem services to human populations and meanwhile are exposed to anthropogenic disturbances. One outcome of human activity is an increasing load in lakes of nutrients such as nitrogen (N), which together with phosphorus has been shown to be the limiting factor for primary production in aquatic ecosystems (1). With a doubling of the rate of nitrogen input into terrestrial environments (2), nitrification coupled with denitrification is of special significance for sustaining the nutrient balance in freshwater lake ecosystems by returning nitrogen into the atmosphere (3).As the first and rate-limiting step of nitrification, production of nitrite from ammonia is potentially driven by ammonia-oxidizing bacteria (AOB), as well as by ammonia monooxygenase-encoding archaea. These putative ammonia-oxidizing archaea (AOA) have recently been proposed to belong to a new phylum, Thaumarchaeota (4). AOB and AOA occur widely in freshwater habitats (5, 6), raising the question of their relative contributions to nitrification (7). The activity of bacterial ammonia oxidizers has been followed by measuring their pop...