The ribosomal-RNA (rRNA) approach to microbial evolution and ecology has become an integral part of environmental microbiology. Based on the patchy conservation of rRNA, oligonucleotide probes can be designed with specificities that range from the species level to the level of phyla or even domains. When these probes are labelled with fluorescent dyes or the enzyme horseradish peroxidase, they can be used to identify single microbial cells directly by fluorescence in situ hybridization. In this Review, we provide an update on the recent methodological improvements that have allowed more reliable quantification of microbial populations in situ in complex environmental samples, with a particular focus on the usefulness of group-specific probes in this era of ever-growing rRNA databases.