Aims: To identify active CO₂-assimilating species of ammonia-oxidizing bacteria in fresh water sediment. Methods and Results: Enrichment cultures were incubated in the presence of ¹³C labelled CO₂, and ¹³C-DNA successfully resolved from ¹²C-DNA by caesium chloride density gradient ultracentrifugation of DNA extracts. Ammonia-oxidizer DNA recovered from these gradients was amplified and characterised by Temporal Temperature Gradient Gel Electrophoresis (TTGE), with confirmatory sequence analysis to identify the metabolically active components of the population. Conclusions: The ¹²C-DNA fraction was dominated by nitrosospiras, in contrast to the ¹³C-DNA fraction which was largely nitrosomonad DNA, in support of the hypothesis that nitrosomonads out-compete nitrosospiras in laboratory culture. Significance and Impact of the Study: The use of stable isotype incorporation into ammonia-oxidizer DNA could therefore circumvent the problems associated with RNA detection to identify metabolically active species in situ.