Like many other parts of microbial ecology, subsurface microbiology has entered the molecular age. As one example of various powerful molecular techniques, fluorescently labeled rRNA-targeted nucleic acid probes today allow an in situ identification of individual microbial cells in their natural habitats. The technique relies on the specific hybridization of the nucleic acid probes to the naturally amplified intracellular rRNA. Fluorescently labeled, rRNA-targeted oligonucleotide probes are perfect tools for many areas of microbial ecology since they can monitor specific populations in environmental samples based on constant genotypic features and not on variable phenotypic features like morphology. In case of immobilized communities like biofilms, exact spatial distributions of microorganisms can be analyzed on a micrometer scale. Recent technical improvements have increased the number of potential applications considerably. Today, better fluorescent dyes enable identification of routinely more than 50% of the cells even in oligotrophic aquatic samples in which the visualization of small cells with low numbers of ribosomes had been problematic. This compares favorably with the usually less than 1% of microorganisms which can be characterized based on cultivation-dependent methods.