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Stable isotope probing (SIP) is a novel technique to characterize structure and in situ function of active microbial populations, which is based on the incorporation of 13C-labelled substrates into nucleic acids. Here, we have traced methylotrophic members of a rice field soil microbial community, which became active upon continuous addition of 13C-methanol (<22 mM) as studied in microcosms. By combining rRNA- and DNA-based SIP, as well as domain-specific real-time PCR detection of templates in fractions of centrifugation gradients, we were able to detect 13C-labelled bacterial rRNA after 6 days of incubation. Fingerprinting and comparative sequence analysis of ‘heavy’ bacterial rRNA showed that mostly members of the Methylobacteriaceae and a novel clade within the Methylophilaceae formed part of the indigenous methylotrophic community. Over time, however, the Methylophilaceae were enriched. Unexpectedly, nucleic acids of eukaryotic origin were detected, mostly in intermediately 13C-labelled gradient fractions. These eukaryotes were identified as fungi mostly related to Fusarium and Aspergillus spp., and also Cercozoa, known as predatory soil flagellates. The detection of fungi and protozoa in 13C-enriched nucleic acid fractions suggests a possible involvement in either direct assimilation of label by the fungi, or a food web, i.e. that primary 13C-methanol consuming methylotrophs were decomposed by fungi and grazed by protozoa.