Microorganisms are responsible for driving the biogeochemical cycling of elements on Earth. Despite their importance and vast diversity, the taxonomic identity of the microorganisms involved in any specific process has usually been confined to that small fraction of the microbiota that has been isolated and cultivated. The recent coupling of molecular biological methods with stable-isotope abundance in biomarkers has provided a cultivation-independent means of linking the identity of bacteria with their function in the environment. Here we show that 13C-DNA, produced during the growth of metabolically distinct microbial groups on a 13C-enriched carbon source, can be resolved from 12C-DNA by density-gradient centrifugation. DNA isolated from the target group of microorganisms can be characterized taxonomically and functionally by gene probing and sequence analysis. Application of this technique to investigate methanol-utilizing microorganisms in soil demonstrated the involvement of members of two phylogenetically distinct groups of eubacteria; the alpha-proteobacterial and Acidobacterium lineages. Stable-isotope probing thus offers a powerful new technique for identifying microorganisms that are actively involved in specific metabolic processes under conditions which approach those occurring in situ.