Stable isotopes in fruit bodies from field studies have been used to infer ectomycorrhizal or saprotrophic status and to understand carbon and nitrogen use, but few controlled culture studies have correlated source and fungal isotopic patterns. Here, we measured natural abundances of 15N and 13C in ten strains of ectomycorrhizal fungi and seven strains of saprotrophic fungi grown on agar with three different primary carbon sources: glucose, glucose plus malt extract, and potato dextrose agar. Eight fungal strains were also grown using position-specific, 13C-labelled glucose (C-1 through C-6 labelled). Most fungi resembled nitrogen sources in δ15N, suggesting that growth on agar media minimizes isotopic fractionation on uptake compared to growth on liquid media, and that in general saprotrophic and mycorrhizal fungi process nitrogen similarly. Saprotrophic fungi were more depleted in 13C than ectomycorrhizal fungi on all media, presumably because of assimilation of 13C-depleted, agar-derived carbon. Results on 13C-enriched glucose indicated that saprotrophic fungi obtained up to 45% of their carbon from the agar substrate. Fungi generally incorporated the individual carbon atoms of glucose in the order, C-4<C-1<C-2, C-3, C-5<C-6, ranging from a mean of 9% for the C-4 atom to 21% for the C-6 atom. Based on these incorporation patterns and intramolecular 13C patterns within glucose, differential incorporation of carbon atoms within glucose among fungal taxa contributed less than 1‰ to isotopic differences among taxa, whereas isotopic fractionation among taxa during metabolism varied up to 4‰. Parallel studies of 13C-enriched and natural abundance substrates were crucial to interpreting our results.