Lichens are generally considered as mutualisms between fungi and green algae or cyanobacteria. These partnerships allow light-exposed and long-living joint structures. The unique organization of lichens provides still unexplored environments for microbial communities. To study lichen-associated bacterial communities, we analyze samples, by a polyphasic approach, from three lichen species (Cladonia arbuscula, Lecanora polytropa and Umbilicaria cylindrica) from alpine environments. Our results indicate that bacteria can form highly structured, biofilm-like assemblages on fungal surfaces and reach considerable abundances of up to 10(8) cells per gram fresh weight. Fluorescence in situ hybridization reveals the predominance of Alphaproteobacteria. Microbial fingerprints performed by PCR-single-strand conformation polymorphism analysis using universal and group-specific primers show distinct patterns for each lichen species. Characterization of cultivable strains and presence of functional genes in the total fraction suggest the involvement of associated bacteria in nutrient cycling. Ubiquitous nifH genes, which encode the nitrogenase reductase, show a high diversity and are assigned to Alphaproteobacteria and Firmicutes, for example, Paenibacillus. Cultivable strains mainly belonging to the genera Acinetobacter, Bacillus, Burkholderia, Methylobacterium and Paenibacillus show lytic (chitinolytic, glucanolytic, and proteolytic) activities, hormone production (indole-3-acetic acid) as well as phosphate mobilization and antagonistic activity toward other microorganisms. The traditional concept of lichens has to be expanded to consider multiple bacterial partners.