Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes is a well established technique for identifying populations of microorganisms at various taxonomic levels in natural and engineered systems. The strength of this technique over alternative nucleic acid technologies is that it can rapidly and specifically yield the number and spatial distribution of microorganisms in complex matrices. When coupled with microautoradiography (MAR-FISH), this technique offers powerful insight into the microbial community structure and their functional activity. In this review we discuss how MAR-FISH can be used to quantify the specific microbial phylotype(s) responsible for a microbially catalyzed process. An example of how both FISH and MAR-FISH were successfully used to quantify phenanthrene- and naphthalene-degrading Proteobacteria and Actinobacteria in coal-tar impacted aquifer sediments at a former manufactured gas plant site is presented. Other applications of this technique are reviewed along with the necessary adaptations for use in soils or sediments. The capabilities and limitations of this technique are compared to alternative technologies for linking microbial community structure and function.