The contribution of bacteria and fungi to NH4+ and organic N (Norg) oxidation was determined in a grassland soil (pH 6.3) by using the general bacterial inhibitor streptomycin or the fungal inhibitor cycloheximide in a laboratory incubation study at 20°C. Each inhibitor was applied at a rate of 3 mg g−1 oven-dry soil. The size and enrichment of the mineral N pools from differentially (NH415NO3 and 15NH4NO3) and doubly labelled (15NH415NO3) NH4NO3 were measured at 3, 6, 12, 24, 48, 72, 96 and 120 hours after N addition. Labelled N was applied to each treatment, to supply NH4+-N and NO3−-N at 3.15 μmol N g−1 oven-dry soil. The N treatments were enriched to 60 atom % excess in 15N and acetate was added at 100 μmol C g−1 oven-dry soil, to provide a readily available carbon source. The oxidation rates of NH4+ and Norg were analysed separately for each inhibitor treatment with a 15N tracing model. In the absence of inhibitors, the rates of NH4+ oxidation and organic N oxidation were 0.0045 μmol N g−1 hour−1 and 0.0023 μmol N g−1 hour−1, respectively. Streptomycin had no effect on nitrification but cycloheximide inhibited the oxidation of NH4+ by 89% and the oxidation of organic N by more than 30%. The current study provides evidence to suggest that nitrification in grassland soil is carried out by fungi and that they can simultaneously oxidize NH4+ and organic N.