To provide insight into the phylogenetic bacterial diversity of the freshwater sponge Spongilla lacustris, a 16S rRNA gene libraries were constructed from sponge tissues and from lake water. Restriction fragment length polymorphism (RFLP) analysis of >190 freshwater sponge-derived clones resulted in six major restriction patterns, from which 45 clones were chosen for sequencing. The resulting sequences were affiliated with the Alpha proteobacteria (n=19), the Actinobacteria (n=15), the Beta proteobacteria (n=2), and the Chloroflexi (n=2) lineages. About half of the sequences belonged to previously described actinobacterial (hgc-I) and beta proteobacterial (beta-II) sequence clusters of freshwater bacteria that were also present in the lake water 16S rRNA gene library. At least two novel, deeply rooting alpha proteobacterial lineages were recovered from S. lacustris that showed <89% sequence similarity to known phylogenetic groups. Electron microscopical observations revealed that digested bacterial remnants were contained within food vacuoles of sponge archaeocytes, whereas the extracellular matrix was virtually free of bacteria. This study is the first molecular diversity study of a freshwater sponge and adds to a growing database on the diversity and community composition of sponge-associated microbial consortia.