Eight species of ectomycorrhizal (ECM) fungi in the genera Amanita, Gymnoboletus, Lactarius, and Russula were isolated from subtropical plant communities in eastern Australia. Two species were isolated from each of rainforest, Nothofagus forest, Eucalyptus forest, and Eucalyptus dominated wallum (heath) forest. These communities differ strongly in their soluble soil nitrogen (N) composition. The ability of the fungi to use inorganic (nitrate, ammonium) and organic (amide, peptide, protein) nitrogen sources was determined. As the fungi did not grow in liquid culture, a ‘floating culture’ technique was devised that allows hyphal growth on a screen floating on liquid medium. With some exceptions, fungal biomass production in floating culture closely reflected fungal growth on solid media assessed by total colony glucosamine content. Most isolates grown in floating culture had similar glucosamine concentrations on all N sources, with isolate specific concentrations ranging from 6 to 12 μg glucosamine g−1 DW. However, Russula spp. had up to 1·7-fold higher glucosamine concentrations when growing with glutamine or ammonium compared to nitrate, glutathione or protein. Floating cultures supplied with 0·5, 1·5, 4·5, or 10 mM N mostly produced greatest biomass with 4·5 mM N. In vitro nitrate reductase activity (NRA) ranged from very low (0·03 μmol NO2 g−1 fw h−1) in Russula sp. (wallum) to high (2·16 μmol NO2 g−1 fw h−1) in Gymnoboletus sp. (rainforest) and mirrored the fungi's ability to use nitrate as a N source. All Russula spp. (wallum, Nothofagus and Eucalyptus forests), Lactarius sp. (rainforest) and Amanita sp. (wallum) utilized ammonium and glutamine but had little ability to use other N sources. In contrast, Amanita species (Nothofagus and Eucalyptus forests) grew on all N sources but produced most biomass with ammonium and glutamine. Only Gymnoboletus sp. (rainforest) showed similar growth with nitrate and ammonium as N sources. Fungal N source use was not associated with taxonomic groups, but is discussed in the context of soil N sources in the different habitats.