The identification of phylogenetic clusters of bacteria that are common in freshwater has provided a basis for probe design to target important freshwater groups. We present a set of 16S ribosomal RNA gene-based oligonucleotide probes specific for 15 of these freshwater clusters. The probes were applied in reverse line blot hybridization, a simple method that enables the rapid screening of PCR products from many samples against an array of probes. The optimized assay was made stringent to discriminate at approximately the singlemismatch level. This made 10 of the probes highly specific, with at least two mismatches to the closest noncluster member in the global database. Screening of PCR products from bacterioplankton of 81 diverse lakes from Belgium, The Netherlands, Denmark, Sweden, and Norway showed that the respective probes were reactive against 5 to 100% of the lake samples. Positive reactivity of six highly specific probes showed that bacteria from actinobacterial clusters ACK-M1 and Sta2-30 and from verrucomicrobial cluster CLO-14 occurred in at least 90% of the investigated lakes. Furthermore, bacteria from alpha-proteobacterial cluster LD12 (closely related to the marine SAR11 cluster), beta-proteobacterial cluster LD28 and cyanobacterial cluster Synechococcus 6b occurred in more than 70% of the lakes. Reverse line blot hybridization is a new tool in microbial ecology that will facilitate research on distribution and habitat specificity of target species at relatively low costs.The introduction of molecular methods into microbial ecology and the selection of rRNA genes as tools for bacterial classification (28,29,35,36) initiated a massive effort to describe bacterial diversity in the environment (15). Random cloning of PCR-amplified genes, the most common procedure for describing diversity, has added thousands of sequences of 16S rRNA genes to the nucleotide databases. These data, which could not have been retrieved through cultivation of bacteria (3,15), are gradually generating a better and more complete view of composition and diversity in several habitats. For instance, it is now clear that both ocean and inland waters have limited bacterial diversity. That is, the evenness in these aquatic systems is relatively low because a few groups of bacteria dominate the systems (10,12,13,23,24,39). Moreover, these dominating groups appear to be widespread over the globe. For freshwater, we recently analyzed all of the available 16S rRNA gene sequences from clone libraries and identified 34 phylogenetic groups that appeared in more than one data set from different lakes or rivers (39). The identification of relatively narrow groups such as these (91 to 99% sequence similarity) creates a starting point for exploration of the functions and ecology of bacteria and allows the design of probes for rapid screening and comparison of bacterial communities in many different freshwater ecosystems.In the present study we describe molecular probes targeting 16S rRNA genes from 15 of the freshwater clusters and explain ...