Environmental microbiology research increasingly focuses on the single microbial cell as the defining entity that drives environmental processes. The inter- actions of individual microbial cells with each other, the environment and with higher organisms shape microbial communities and control the functioning of whole ecosystems. A single-cell view of microorgan- isms in their natural environment requires analytical tools that measure both cell function and chemical speciation at the submicrometre scale. Here we review the technical capabilities and limitations of high-resolution secondary ion mass spectrometry (NanoSIMS) and scanning transmission (soft) X-ray microscopy (STXM) and give examples of their appli- cations. Whereas NanoSIMS can be combined with isotope-labelling, thereby localizing the distribution of cellular activities (e.g. carbon/nitrogen fixation/ turnover), STXM provides information on the location and chemical speciation of metabolites and products of redox reactions. We propose the combined use of both techniques and discuss the technical challenges of their joint application. Both techniques have the potential to enhance our understanding of cellular mechanisms and activities that contribute to microbi- ally mediated processes, such as the biogeochemical cycling of elements, the transformation of contami- nants and the precipitation of mineral phases.