The distribution of the phylogenetically narrow R-BT065 cluster (Betaproteobacteria) was examined in 102 freshwater lakes, reservoirs, and various ponds located in the central Europe (in total 122 samples) by using a cluster-specific FISH probe. These habitats differ markedly in their pH, conductivity, trophic status, surface area, altitude, bedrock type, and other limnological characteristics. Despite of the broad ecological diversity of the investigated habitats, the cluster was detected in 96.7% of the systems, and its occurrence was not restricted to a certain habitat type. However, the relative proportions of the cluster in total bacterioplankton were significantly lower in humic and acidified lakes than in pH-neutral or alkaline habitats. The cluster accounted, on average, for 9.4% of total bacterioplankton (range 0-29%). The relative and absolute abundance of these bacteria were significantly and positively related to increasing pH, conductivity, and proportion of low molecular weight compounds in dissolved organic carbon (DOC), and negatively related to total DOC and dissolved aromatic carbon content. Together, these parameters explained 55.3% of the variability in the occurrence of the cluster. Surprisingly, no clear relationship of the R-BT065 bacteria to factors indicating trophic status of habitats, i.e. different forms of phosphorus and chlorophyll-a content, was found. Based on our results and literature data we conclude that the R-BT065 cluster represents a ubiquitous, highly active segment of bacterioplankton in non-acidic lakes and ponds, and that algal-derived substrates likely form the main pool of substrates responsible for its large growth potential and broad distribution in freshwater habitats.