Summary We have coupled fluorescence in situ hybridization (FISH) with Raman microscopy for simultaneous cultivation-independent identification and determination of 13C incorporation into microbial cells. Highly resolved Raman confocal spectra were generated for individual cells which were grown in minimal medium where the ratio of 13C to 12C content of the sole carbon source was incrementally varied. Cells which were 13C-labelled through anabolic incorporation of the isotope exhibited key red-shifted spectral peaks, the calculated ‘red shift ratio’ (RSR) being highly correlated with the 13C-content of the cells. Subsequently, Raman instrumentation and FISH protocols were optimized to allow combined epifluorescence and Raman imaging of Fluos, Cy3 and Cy5-labelled microbial populations at the single cell level. Cellular 13C-content determinations exhibited good congruence between fresh cells and FISH hybridized cells indicating that spectral peaks, including phenylalanine resonance, which were used to determine 13C-labelling, were preserved during fixation and hybridization. In order to demonstrate the suitability of this technology for structure–function analyses in complex microbial communities, Raman-FISH was deployed to show the importance of Pseudomonas populations during naphthalene degradation in groundwater microcosms. Raman-FISH extends and complements current technologies such as FISH-microautoradiography and stable isotope probing in that it can be applied at the resolution of single cells in complex communities, is quantitative if suitable calibrations are performed, can be used with stable isotopes and has analysis times of typically 1 min per cell.