We analysed the phylogenetic relatedness of 16S rRNA genes from freshwater bacteria affiliated with the class Actinobacteria. A polymerase chain reaction assay was developed to identify reliably rare Actinobacteria-related inserts within 16S rRNA gene clone libraries. In 18 libraries constructed from seven freshwater systems, altogether 63 actinobacterial sequence types were collected from a total of > 1800 clones. Sixty of the newly obtained sequences grouped within four distinct phylogenetic lineages. They constitute approximately 75% of the nearly complete sequences within these clusters that are presently available. A comparison with > 300 sequences from various soil habitats revealed that two of these monophyletic actinobacterial clades (acI and acII) almost exclusively harbour 16S rRNA sequence types from freshwaters and estuaries. This may indicate that such bacteria are not inoculated to freshwaters from terrestrial sources, but are autochthonous components of freshwater microbial assemblages. In contrast, sequence types from freshwaters, marine sediments and soils were clearly mixed in another of the actinobacterial lineages (acIV). Sequence divergence within acIV was the highest of all four lineages (88% minimum similarity), which potentially reflects its radiation across several habitat types. Within the freshwater lineages, groups of essentially identical sequence types were retrieved from geographically distant aquatic systems with strikingly different hydrological and limnological characteristics. This points to the necessity to investigate genotypic variability, in situ abundances and activities of these Actinobacteria in freshwater plankton in greater detail by cultivation-independent techniques.