Abstract We report the first simultaneous measurements of δ 15N and δ 13C of DNA extracted from surface soils. The isotopic composition of DNA differed significantly among nine different soils. The δ 13C and δ 15N of DNA was correlated with δ 13C and δ 15N of soil, respectively, suggesting that the isotopic composition of DNA is strongly influenced by the isotopic composition of soil organic matter. However, in all samples DNA was enriched in 13C relative to soil, indicating microorganisms fractionated C during assimilation or preferentially used 13C enriched substrates. Enrichment of DNA in 15N relative to soil was not consistently observed, but there were significant differences between δ 15N of DNA and δ 15N of soil for three different sites, suggesting microorganisms are fractionating N or preferentially using N substrates at different rates across these contrasting ecosystems. There was a strong linear correlation between δ 15N of DNA and δ 15N of the microbial biomass, which indicated DNA was depleted in 15N relative to the microbial biomass by approximately 3.4‰. Our results show that accurate and precise isotopic measurements of C and N in DNA extracted from the soil are feasible, and that these analyses may provide powerful tools for elucidating C and N cycling processes through soil microorganisms.