Identifying the function of uncultured microbes in their environments today remains one of the main challenges for microbial ecologists. In this article, we describe a new method allowing simultaneous analysis of microbial identity and function. This method is based on the visualization of oligonucleotide probe-conferred hybridization signal in single microbial cells and isotopic measurement using high-resolution ion microprobe (NanoSIMS). In order to characterize the potential of the method, an oligonucleotide containing iodized cytidine was hybridized on fixed cells of Escherichia coli cultured on media containing different levels of 13C or 15N. Iodine signals could clearly be localized on targeted cells and the isotopic enrichment could be monitored at the single-cell level. The applicability of this new technique to the study of in situ ecophysiology of uncultured microorganisms within complex microbial communities is illustrated.